WO2003055379A2 - Method using a surface-selective nonlinear optical technique for imaging of biological samples - Google Patents

Abstract

Nonlinear optical-active species which are analogues of fluorescent labels, markers, tags, contrast agents and targeting constructs used for imaging or detection by a surface-selective nonlinear optical technique are disclosed. A surface-selective nonlinear optical technique, such as second harmonic or sum or difference frequency generation, is used to image biological tissues or cells, in-vivo, in-situ or in-vitro, in the presence of one or more of these species&period

Description

METHOD USING A SURFACE-SELECTIVE NONLINEAR OPTICAL TECHNIQUE FOR IMAGING OF BIOLOGICAL SAMPLES

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims benefit of United States provisional application number

60/342,882, filed on December 24, 2001, which is hereby incorporated by reference herein in its entirety.

1. FIELD OF THE INVENTION

The present invention relates to a method for imaging animal tissues, organs, cells and other animal samples.

2. BACKGROUND OF THE INVENTION

Imaging tissues and biological cells is an important technique in modern biology and medicine. Medical optical imaging - especially in- vivo as with optical biopsies - can be used to identify or diagnose diseased tissues such as malignant tissues. Fluorescence- based imaging is useful but has drawbacks due to background fluorescence and auto- fluorescence of the samples, making it often difficult, for example, to discriminate a diseased tissue from neighboring healthy tissues. Fluorescent markers or tags which are targeted to disease tissues are known in the art and are useful in increasing the signal of a potentially diseased tissue above the background fluorescence present in most samples. Surface-selective nonlinear optical-based imaging, such as that using second harmonic generation, can be useful in further reducing the background and increasing the signal to noise of the imaging technique as found in prior art. Surface-selective nonlinear optical imaging (second harmonic imaging, for example) of animal samples in the past has only relied on the intrinsic nonlinear response of the various tissues, cells, etc. However, the native nonlinear optical cross-section in tissues or cells can be low.

3. SUMMARY OF THE INVENTION

The invention provides a method for imaging a living animal or living cell using a surface-selective nonlinear optical technique. The method comprising the steps of administering to a living animal or living cell one or more selected from the group consisting of a targeting construct, a label, a decorator, an indicator and an enhancer, illuminating the living animal or living cell with one or more light beams at one or more fundamental frequencies, and detecting a nonlinear optical light beam emanating from the living animal or living cell, wherein said the nonlinear optical light beam forms an image of the living animal or living cell.

The invention provides a method of measuring a target site within a living subject. A nonlinear-active contrast agent selected so as to provide a nonlinear optical signal contrast for the target site in vivo when the target site is illuminated with a light source is administered to the living subject. The contrast agent is allowed to achieve sufficient distribution and localization within the body of said living subject. A medical device or instrument optically coupled to a light source and a light detector is used in the performance of a medical or surgical procedure upon the living subject. The target site is illuminated with light from the light source, where the light source selected such that the contrast agent in vivo may interact with the light and generate a nonlinear optical signal. The nonlinear optical signal is detected using a light detector, and a measurable parameter of the target site is determined using the detected light based upon a function of the distribution and localization of the contrast agent. An output signal is generated using the measurable parameter.

The invention provides a method for in vivo identification of tumor tissue associated with a disease state in a living subject. The method comprises the steps of administering to the living subject having directly viewable tumor tissue a diagnostically effective amount of at least one biologically compatible nonlinear-active targeting construct comprising a tumor-avid moiety, so as to allow the nonlinear-active targeting construct to bind to and/or be taken up selectively in vivo by the directly viewable tumor tissue, illuminating an in vivo body part of the living subject comprising the directly viewable tumor tissue with a light beam having at least one wavelength in the nonlinear optical response spectrum of the nonlinear-active targeting construct, and detecting a nonlinear-optical light beam emanating from any targeting construct bound to or taken up by the directly viewable tumor tissue, wherein the nonlinear light beam indicates the location and/or surface area of the tumor tissue in the body part.

These and other objects and features of the invention will be more fully appreciated when the following detailed description of the invention is read in conjunction with the accompanying drawings. 4. BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 depicts one embodiment of the apparatus in which the mode of generation and collection of the second harmonic light is by reflection off the sample.

Fig. 2 depicts one embodiment of an apparatus in which the mode of generation and collection of the second harmonic light is by total internal reflection through a prism. The prism is coupled by an index-matching material to a sample.

Fig. 3 depicts one embodiment of an apparatus in which the mode of generation and collection of the second harmonic light is by total internal reflection through a wave-guide with multiple reflections as denoted by the dashed line inside the wave-guide. Fig. 4 depicts one embodiment of a flow-cell for delivery and removal of biological components and other fluids to the sample.

Fig. 5 depicts three embodiments of an apparatus in which the mode of generation and collection of the second harmonic light is by transmission through a sample. In Fig. 5A, the second harmonic beam is co-linear with the fundamental. In Fig. 5B, the second harmonic is collected from a direction orthogonal to the fundamental ('right-angle collection'). In Fig. 5C, the second harmonic light is collected by an integrating sphere and a fiber optic line.

Fig. 6 depicts an embodiment of the transformation, using a series of optical components, of a collimated beam of the fundamental light into a line shape suitable for scanning a substrate.

Fig. 7 depicts an embodiment of an apparatus in which the mode of generation and collection of the second harmonic light is through a fiber optic. Fig. 7 A depicts the use of a bundle of fiber optic lines and Fig. 7B depicts the use of beads coupled to the end of a fiber for attaching probes. Fig. 8 depicts three embodiments of an apparatus in which the mode of generation and collection of the second harmonic light is by transmission through a sample. Fig. 8 A depicts both the fundamental and second harmonic beams travelling co-linearly through a sample. Fig. 8B depicts the fundamental and second harmonic beams being refracted at the top surface (top surface contains attached probes) of a substrate with this surface generating the second harmonic light. Fig 8C depicts a similar apparatus to Fig. 8B except that the bottom surface (bottom surface contains attached probes) generates the second harmonic light.

Fig. 9 depicts two embodiments of an apparatus in which second harmonic light is generated by total internal reflection at an interface. The points of generation of the second harmonic light are denoted by the circles, hi Fig. 9 A, a dove prism is used to guide the light to a surface capable of generating the second harmonic light (bottom surface of prism but can also be another surface coupled to the prism through an index-matching material), hi Fig. 9B, a wave-guide structure is used to produce multiple points of second harmonic generation. Fig. 10 depicts three embodiments of an apparatus in which second harmonic light is generated using a fiber optic line (with attached probes at the end of the fiber). Fig. 10A depicts an apparatus in which both generation and collection of the second harmonic light occur in the same fiber. Fig. 10B depicts the use of a bead containing surface-attached probes at the end of the fiber. Fig. IOC depicts an apparatus in which the second harmonic light is generated at the end of the fiber optic (containing attached probes) and collected using a mirror or lens external to the fiber optic.

Fig. 11 depicts two embodiments of an apparatus using an optical cavity for power build-up of the fundamental.

Fig. 12 depicts three embodiments of an apparatus in which the mode of generation and collection of the second harmonic light uses reflection of the light from an interface.

5. DESCRIPTION OF THE INVENTION

The present invention discloses the use of exogenous molecular tools to enhance the nonlinear optical signal from an animal sample, such as tissues, cells or organs, which is imaged using a surface-selective nonlinear optical technique. Four tools are disclosed herein. The first are nonlinear optical labels (nonlinear-active moieties, molecules or particles which attach specifically to some molecule of interest - target to some specific site, for example a specific cellular membrane receptors in a tissue sample. The second class of tools are decorators which are molecules which themselves are labeled with a nonlinear-active label and which have some chemical affinity and specificity for a third molecule to be decorated. An antibody, with a specific chemical affinity to some cell receptor to be decorated, that is labeled with a nonlinear active label is an example of a decorator. In the fluorescence-based imaging art fluorescent labels or decorators are also referred to as 'contrast agents', 'conjugates', 'tags', 'markers' and 'immunoconjugates'. Third are indicators which are nonlinear-active molecules or particles that sense the electric field, potential or charge density near some interface of interest - in this case, the surface of tissues, cells or other biological samples that are imaged. Indicators work by orienting themselves in response to an electric charge density on a surface - and thus, through the coherence effect of surface-selective nonlinear optical techniques - modulate the intensity or wavelength (and therefore frequency) of the measured nonlinear optical radiation as the electric properties of the cell or tissue surface is changed by another agent such as a drug, ligand or agonist. Fourth are resonance enhancers which serve to increase the cross-section of the nonlinear optical response of labels, decorators or indicators when they are in proximity to the latter; this enhancement of the nonlinear optical response can occur through such well known resonance-enhancing chemical or electromagnetic coupling mechanisms. Resonance enhancers can be attached to labels, decorators or indicators or allowed to come into proximity with the latter, for example by adsorbing or attaching resonance enhancers independently to cell surfaces, or simply by suspending tissues or cells in a medium containing a concentration of the enhancers. Hereafter, the particular aforementioned tool or combination of tools used for surface-selective nonlinear optical imaging of animal tissues, cells, organs, etc. will be referred to as a 'nonlinear optical contrast agent'.

Not every physical or pathological state has an inherent optical signal or a signal that can be readily discriminated from the background. For instance, many of the properties that are associated with tumors are bulk phenomena that may not apply to very small tumors. In these instances, nonlinear optical labels (also referred to as 'contrast agents' in the art) will help when the native signal is weak, absent, or non-specific. Contrast agents can be directed toward specific tissues, including antibodies against service receptors, which are uniquely expressed on tumors. The goal of contrast, for example, is to give the physician or surgeon the same advantages in-vivo that the pathologist has ex-vivo. New methods of targeting (e.g., receptor targeting), have helped improve contrast, hi addition, antibodies and antibody fragments in the (fluorescence-based) contrast agent art are getting more and more specific. Optical contrast: has several advantages: it is targetable, tolerable, safe, highly sensitive, inexpensive, not radioactive, and has direct placement for many applications in the radiotracer market. Optical imaging allows better real-time detection than both PET and PCR because it is in-vivo and real-time. Examples of some contrast agents that are being developed for fluorescence-based optical imaging include a prostate antigen targeted against an extracellular region of the PSMA molecule, a lymphatic marker that biodistributes, and a colon marker. In general, every technique for the use of a fluorescent contrast agent, conjugate, or marker in fluorescence-based optical imaging of cells, tissues, organs, etc. can be used directly with the analogous nonlinear optical contrast agent, conjugate or marker for surface-selective nonlinear optical imaging. Selection of the appropriate agent, conjugate or marker is straightforward and based on the specific sample (e.g., tissue, cells) to be imaged. For example, tumorous tissue will display certain proteins on cell surfaces and specific markers for these receptors will discriminate these tissues from non-tumorous ones. The markers can be conjugated to a nonlinear active label or decorated with a labeled antibody, for example using techniques well known to one skilled in the art. Detection and imaging of the sample is then accomplished by imaging techniques known in the art. The method and apparatus of surface-selective nonlinear optical imaging is well known. The following (and references therein) are exemplary of the prior art in nonlinear optical and fluorescence- based biological imaging:

U.S. Pat. No. 6,217,847, April, 2001, Contag et al./U.S. Pat. No. 6,280,386 Aug., 2001, Alfano et al./U.S. Pat. No. 6,208,88 Mar., 2001,Alfano et al./U.S. Pat. No. 5,853,370, Chance et al. U.S. Pat. No. 4,821,117 April 1989, Sekiguchi et al./U.S. Pat. No. 5,318,023 June 1994 Vari et al./U.S. Pat. No. 5,408,996 April 1995 Salb et al./U.S. Pat. No. 5,650,135 July 1997 Contag et al./U.S. Pat. No. 5,677,199 October 1997 Arrhenuis/U.S. Pat. No. 5,807,261 September 1998 Benaron et al./U.S. Pat. No. 6,167,297 December 2000 Benaron et al./U.S. Pat. No. 6,217,847 April 2001 Contag et al./U.S. Pat. No. 6,246,901 June 2001 Benaron/U.S. Pat. No. 6,284,223 September 2001 Luiken/Y. Guo et al., "Subsurface tumor progression investigated by noninvasive optical second harmonic tomography". The present invention therefore serves as a bridge between, on the one hand, the optical imaging techniques with fluorescent conjugates, tags, contrast agents and markers and fluorescence imaging in animal tissues, cells, organs, etc. with these; and surface-selective nonlinear optical imaging of animal samples, on the other hand, which has not in the prior art employed the use of nonlinear active labels, decorators, indicators or enhancers. A general description and a specific example of nonlinear optical labels is found in: '"SHG Labels' for Detection of Molecules by Second Harmonic Generation", J.S. Salafsky, Chemical Physics Letters, 2001, v.342, N5-6, 20 July.

Delivery of the labels, indicators, decorators or enhancers (or conjugates and/or some combination thereof) to the tissues in-vivo can be accomplished by any of the techniques known in the art for fluorescent, radioactive, NMR or other contrast agent or conjugate for the corresponding detection technique (e.g., radiodetection, fluorescence, NMR, etc.). For example, the delivery can be via intravenous injection, adminstered orally or through inhalation, or suspension of tissue, cells, organs, etc. in a solution of the labels, indicators, decorators or enhancers.

A resonance-enhancing or surface-enhancing (hereafter an "enhancer") species can be placed near to, or attached to, a molecule, surface or particle to enhance the nonlinear active cross-section (the second order nonlinear) of the sample for an increased signal in detection by a surface-selective nonlinear optical technique. The enhancing species increases the cross-section of the nonlinear-active species through electromagnetic or chemical coupling between the resonance-enhancing species and the nonlinear-active species. For example, the electromagnetic or chemical coupling between the nonlinear- active moiety and the enhancer leads to an increased cross-section for generation of second harmonic light (or sum or difference-frequency light) due to a strong electronic transition or plasmon resonance of the enhancer (through, for example, a local-field effect).

The following (and references therein) are exemplary of the prior art in the production, design and use of resonance-enhancing particles for nonlinear optical processes (SHG, Surface-enhanced resonance raman, etc.):

S. Nie and S. Emory, Science, 1997, 75, 1102; P.V. Kamat, M. Flumiani, G.N. Hartland, J. Phys. Chem. B, 1998, 102, 3123;

H. Ditlbacher et al., Appl. Phys. B, 2001, DOI: 10, 1007/s003400100700;

C. Sonnichsen et al., Appl. Phys. Lett., 2000, 77, 2949;

B. Lamprecht et al., Appl. Phys. B, 1997, 64, 269;

J.P. Novak, J. Am. Chem. Soc, 2000, 122, 12029; S.R. Emory, W.E. Haskins, S.Nie, J. Am. Chem. Soc. 1998, 120, 8009;

W.P. McConnell et al, J. Phys. Chem. B 2000, 104, 8925; F.W. Nance, B.I. Lemon, J.T. Hupp, J. Phys. Chem. B 1998, 102, 10091;

P. Galletto et al., J. Phys. Chem. B 1999, 103, 8706;

S.R. Emory, S. Νie, J. Phys. Chem. B 1998, 102, 493.

Examples of resonance-enhancing species in the art are the following: metal or metallic (e.g., gold and silver) nanoparticles or colloidal particles, metal-coated particles (e.g., silver-coated latex nanospheres), aggregates or clusters of any of the aforementioned, rationally-designed clusters, chains or aggreates of the aforementioned (e.g., for symmetry- breaking: non-centrosymmetric aggregates, particles or clusters), etc.

In many cases, the experimenter will decide to use labels in a measurement. In this case, the selection of a known nonlinear active species is straightforward and the choice depends mainly on the wavelength (and therefore frequency) responsivity of the species, the presence of any fluorescence background, and the wavelength (and therefore frequency) of the fundamental beam at hand; alternatively, new candidate species for nonlinear activity can be readily tested for activity using means well known to one skilled in the art (e.g. using an air- water interface or an EFISH measurement). Once a selection is made, the species can be coupled to the target of interest (e.g., a protein, an antibody, etc.) using techniques well known to those skilled in the art. Then the labeled conjugates can be used to image samples using a surface-selective nonlinear optical technique. In alternate embodiments of the invention, at least two distinguishable nonlinear-active labels are used. Where possible, the orientation of the attached two or more distinguishable labels could also be chosen to facilitate well-defined directions of the emanating coherent nonlinear light beam. The two or more distinguishable labels can be used in where multiple fundamental light beams at one or more frequencies, incident with one or more polarization directions relative to the living animal or living cell, are used, with the resulting emanation of at least two nonlinear light beams.

Indicators can be used instead of labels. The choice of an indicator can be readily made by testing for probe-target binding in the presence and absence of an indicator while measuring the nonlinear optical radiation from the surface. Indicators are added directly to the medium containing the sample. An experimenter can simply measure the nonlinear optical radiation before, during or after adding the indicator and compare measurements of the sample in the presence and absence of the indicator. Many nonlinear active molecules can be used as an indicator and selection of the appropriate one will be governed mainly by wavelength considerations - the presence of background fluorescence, for example, the wavelength (and therefore the frequency) of the fundamental beam to be used. One skilled in the art can readily select the appropriate indicator for the specific experiment at hand. 4PyMPO-MeMs is an excellent indicator and responds readily to changes in surface charge density that occur as targets bind to the surface-attached probes.

Enhancers or decorators can be used in the experiment as well and their use will be guided by the specific requirements of each experiment. Enhancers can be used to greatly enhance the nonlinear optical activity of labels (to increase the nonlinear hypeφolarizability of a label) and one skilled in the art can readily attach or bring into proximity the enhancers to the labels to achieve this enhancement.

One skilled in the art will be able to readily design the appropriate combination and use of indicators, labels, enhancers or decorators for each experiment at hand and according to the specific requirements of each experiment.

Nonlinear optical contrast agents - for example, as immunospecific second- harmonic active molecules or particles - can be used for second harmonic imaging studies of cells, membranes, tissues, involving, for example, microscopy or confocal microscopy, and be imaged in-vitro, in-situ or in-vivo. For in-vivo applications, the labels can be delivered to the sample of interest by well known techniques that use fluorescent dyes for imaging or tracing and, for example, endoscopes. In-vitro applications of the present invention include diagnosis or identification of tissue samples, for example as used in histology or histopathology.

A wide degree of flexibility is expected in the design of the apparatus including, but not limited to, the source of the fundamental light, the optical train necessary to control, focus or direct the fundamental and nonlinear light beams, the design of the array, the detection system, and the use of a grating or filters and collection optics. The term fundamental beam as used herein refers to an illuminating light at a given frequency fundamental. The term nonlinear optical beam refers to second harmonic frequency light, or sum frequency or difference frequency light. Where embodiments are described for second harmonic light (i.e., nonlinear optical light generated at twice the frequency of the illuminating light 2ω), sum or difference frequency nonlinear optical light can also be used (i.e., nonlinear optical light generated at the sum or difference of frequencies of illuminating light coi and ω₂). The terms beam and light are used interchangeably. Values of wavelength are preferably between 100 - 3000 nanometers (and similarly for corresponding frequencies of light). The mode of generation (irradiation) or collection can be varied including, for example, the use of evanescent wave (total internal reflection), planar wave guide, reflection, or transmission geometries, fiber-optic, near-field illumination, confocal techniques or the use of a microcavity or integrating detection system. A number of methods for scanning a microarray on a solid surface are described. Examples include U.S. Pat. No.'s Trulson et al. (1998), Trulson et al. (2000), Stern et al. (1997) and Sampas (2000)- relevant portions of which are incorporated by reference herein. Fiber optic lines can be especially useful for minimally invasive in-vivo imaging of tissues and cells. A surface-selective nonlinear optical microscope can be employed in the imaging process, for example a second harmonic generation microscope as is found in the art. The polarization of the fundamental and nonlinear beams can be selected with polarizing optics elements to optimize the imaging signal and reduce background radiation.

Localization Of the Nonlinear Optical Contrast Agents in-vivo:

hi the case of "targeted" entities, that is, entities which contain a targeting moiety~a molecule or feature designed to localize the entity within a subject or animal at a particular site or sites, localization refers to a state when an equilibrium between bound, "localized", and unbound, "free" entities within a subject has been essentially achieved. The rate at which such an equilibrium is achieved depends upon the route of administration. For example, a conjugate or contrast agent administered by intravenous injection to localize thrombi may achieve localization, or accumulation at the thrombi, within minutes of injection. On the other hand, a conjugate administered orally to localize an infection in the intestine may take hours to achieve localization.

Alternatively, localization may simply refer to the location of the entity within the subject or animal at selected time periods after the entity is administered. For example, in experiments detailed herein, Salmonella are administered (e.g., orally) and their spread is followed as a function of time. In this case, the entity can be "localized" immediately following the oral introduction, inasmuch as it marks the initial location of the administered bacteria, and its subsequent spread or recession (also "localization") may be followed by imaging. In a related aspect, localization of, for example, injected tumors cells expressing a light-generating moiety, may consist of the cells colonizing a site within the animal and forming a tumor mass.

By way of another example, localization is achieved when an entity becomes distributed following administration. For example, in the case of a conjugate administered to measure the oxygen concentration in various organs throughout the subject or animal, the conjugate becomes "localized", or informative, when it has achieved an essentially steady- state of distribution in the subject or animal.

In all of the above cases, a reasonable estimate of the time to achieve localization may be made by one skilled in the art. Furthermore, the state of localization as a function of time may be followed by imaging the nonlinear-optical active conjugate according to the methods of the invention.

Examples of tissues, organs or animal cells to be imaged

(either in-vivo or in-vitro include lungs, liver, brain, heart, kidney, etc. It is generally good practice to allow the nonlinear-active targeting construct to bind to and/or be taken up by any targeting tissue that may be present at the site under investigation and then, before administration of the supplemental fluorescing targeting construct(s), to substantially remove (e.g., wash) from the body part any unbound targeting. Usually, supplemental targeting constructs are successively administered to build up the nonlinear optical signal from the target tissue. For example, if the nonlinear-active targeting construct comprises a humanized IgG monoclonal antibody specific for a breast cancer antigen conjugated to a nonlinear-active dye (such as the amine-reactive oxazole dye (SE) 1 -(3-(succinimidyloxycarbonyl) benzyl)-4-(5-(4-methoxyphenyl) oxazol-2-yl)pyridinium bromide (PyMPO, SE: Molecular Probes Corp.) the next-administered targeting construct may comprise an anti-nonlinear-active label antibody. Those of skill in the art will be able to devise combinations of successively administered targeting constructs, each of which specifically binds to the targeting construct or to one or more of the earlier administered supplemental targeting constructs.

The nonlinear-active moiety of the targeting construct or of the supplemental targeting ligand(s) can be any chemical or protein moiety that is biologically compatible

(e.g., suitable for in vivo administration). Since the targeting ligand is administered to living tissue, biological compatibility includes the lack of substantial toxic effect to the individual in general, and to the target tissue, in particular. Toxicity of useful targeting constructs can be determined using animal studies as known in the art.

Preferably, the targeting construct (e.g., the ligand moiety of the invention targeting construct) is selected to bind to and/or be taken up specifically by the target tissue of interest, for example to an antigen or other surface feature contained on or within a cell that characterizes a disease or abnormal state in the target tissue. As in other diagnostic assays, it is desirable for the targeting construct to bind to or be taken up by the target tissue selectively or to an antigen associated with the disease or abnormal state; however, targeting constructs containing ligand moieties that also bind to or are taken up by healthy tissue or cell structures can be used in the practice of the invention method so long as the concentration of the antigen in the target tissue or the affinity of the targeting construct for the target tissue is sufficiently greater than for healthy tissue in the field of vision so that an image representing the target tissue. For example, colon cancer is often characterized by the presence of carcinoembryonic antigen (CEA), yet this antigen is also associated with certain tissues in healthy individuals. However, the concentration of CEA in cancerous colon tissue is often greater than is found in healthy tissue, so an anti-CEA antibody could be used as a ligand moiety in the practice of the invention. In another example, deoxyglucose is taken up and utilized by healthy tissue to varying degress, yet its metabolism in healthy tissues, except for certain known organs, such as the heart, is substantially lower than in tumor. The known pattern of deoxyglucose consumption in the body can therefore be used to aid in determination of those areas wherein unexpectedly high uptake of deoxyglucose signals the presence of tumor cells.

In one embodiment according to the present invention, the disease or abnormal state detected by the invention method can be any type characterized by the presence of a known target tissue for which a specific binding ligand is known. For example, various heart conditions are characterized by production of necrotic or ischemic tissue or production of artherosclerotic tissue for which specific binding ligands are known. As another illustrative example, breast cancer is characterized by the production of cancerous tissue identified by monoclonal antibodies to CA15-3, CA19-9, CEA, or HER2/neu. It is contemplated that the target tissue may be characterized by cells that produce either a surface antigen for which a binding ligand is known, or an intracellular marker (i.e. antigen), since many targeting constructs penetrate the cell membrane. Representative disease states that can be identified using the invention method include such various conditions as different types of tumors, bacterial, fungal and viral infections, and the like. As used herein "abnormal" tissue includes precancerous conditions, necrotic or ischemic tissue, and tissue associated with connective tissue diseases, and auto-immune disorders, and the like. Examples of the types of target tissue suitable for examination using the invention method include cardiac, breast, ovarian, uterine, lung, endothelial, vascular, gastro-intestinal, colorectal, prostatic tissue, endocrine tissue, and the like, as well as combinations of any two or more thereof.

The present invention provides method(s) for in vivo identification of a biological sample (e.g., tissues, cells, organs in-vivo, in-situ or in-vitro) associated with a disease state in a subject in need thereof. The invention method(s) comprise administering to the subject a diagnostically effective amount of a nonlinear-active targeting constmct comprising a tumor-avid compound so as to allow the targeting construct to bind to and/or be taken up by the target tumor tissue, irradiating a body part of the subject suspected of containing the target tumor tissue and viewing or imaging the nonlinear optical radiation emanating from the targeting construct bound to the target tumor tissue so as to determine the location and/or surface area of the target tumor tissue in the body part. Tumor-avid compounds that are preferentially "taken up" by tumor cells and can be used as the ligand moiety in the invention targeting constructs generally enter the cells through surface or nuclear receptors, such as hormone receptors, or through pores, hydrophilic "windows" in the cell lipid bilayer, and the like. If the putative body part is a body opening of the subject or a surgically produced interior site, an endoscopic device is optionally used to direct the fundamental light to the body part and to receive the nonlinear optical light from the targeting construct for direct viewing or photodetection. Thus, an endoscopic device can aid in detection of tumor tissue associated with a disease state by direct viewing of nonlinear light emanating from the tumor-avid ligand attached to or taken up by the target tumor tissue.

In an embodiment of the invention method, the nonlinear-active targeting construct comprises a biologically compatible nonlinear-active species and a tumor-avid ligand moiety that preponderantly binds to and/or is taken up by tumor tissue, such as deoxyglucose, somatostatin, a somatostatin receptor-binding peptide, methionine, and the like. Additional tumor-avid ligands are hormones and other compounds which bind to and/or are taken up preferentially by tumor cells, for example by receptors, such as nuclear receptors, expressed by tumor cells. The tumor can be any type for which a specific tumor- avid ligand is known or can be pre-determined using screening procedures described herein. The invention method is suited to in vivo detection of tumor tissue located at an interior site in the subject, such as within a natural body cavity or a surgically created opening, without the need for an endoscopic device. For example, the tumor tissue can be contemporaneously viewed through a surgical opening to facilitate a procedure of biopsy or surgical excision. As the precise location and/or surface area of the tumor tissue are readily determined by the invention diagnostic procedure, the invention method is a valuable guide to the surgeon, who needs to "see" the exact outlines, size, etc. of the mass to be resected as the surgery proceeds.

5.1 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS hi a specific embodiment, the invention includes a method for detecting the localization of a biocompatible entity in an animal subject. The entity can be a molecule, macromolecule, cell, microorganism (including a pathogen), a particle, or the like.

The method includes administering to the subject a nonlinear-active species (and/or an enhancer). The species may be conjugated to another species which itself is specific for a tissue, molecule, cell, etc. of the biological sample to be imaged, by a variety of techniques, including incorporation during synthesis of the entity (e.g. chemical or genetic, such a fusion protein of an antibody fragment and a light-generating protein), chemical coupling post-synthesis, non-covalent association, in-situ synthesis in the entity (e.g. expression of a protein in a transformed cell), or in situ activatable promoter-controlled expression of a protein in cells of a transgenic animal stimulated by a promoter inducer (e.g. interferon-activated promoter stimulated by infection with a virus). The administering step for the fusion protein comprises introducing a nucleic acid encoding a fusion protein comprising a label such that the fusion protein is expressed by the living animal or living cell. The fusion protein can comprise bacteriorhodopsin, green fluorescent protein, or a genetic mutant thereof.

After a period of time in which the conjugate can localize in the subject, the subject is immobilized within the detection field of a photodetector device for a period of time effective to measure a sufficient amount of photon scattering (due to irradiation with a fundamental light beam) with the photodetector device to construct an image. An exemplary photodetector device is an intensified charge-coupled device (ICCD) camera coupled to an image processor. If the image can be constructed in a time short relative to the time scale at which an "unimmobilized" subject moves, the subject is inherently "immobilized" during imaging and no special immobilization precautions are required. An image from the photon emission data is then constructed.

The method can be used in a number of specific applications, by attaching, conjugating or incorporating targeting moieties onto the entity. The targeting moiety may be an inherent property of the entity (e.g. antibody or antibody fragment), or it may be conjugated to, attached to, or incorporated in the entity (e.g. liposomes containing antibodies). Examples of targeting moieties include antibodies, antibody fragments, enzyme inhibitors, receptor-binding molecules, various toxins and the like. Targets of the targeting moiety may include sites of inflammation, infection, thrombotic plaques, cell surface- expressed molecules and tumor cells. Markers distinguishing these targets, suitable for recognition by targeting moieties, are well known.

Further, the method may be used to detect and localize sites of infection by a pathogen in an animal model, using the pathogen (e.g. Salmonella) conjugated to a light- generating moiety as the entity. In a related embodiment, the invention includes a noninvasive method for detecting the level of a biocompatible entity in an animal subject over time. The method is similar to methods described above, but is designed to detect changes in the level of the entity in the subject over time, without necessarily localizing the entity in the form of an image. This method is particularly useful for monitoring the effects of a therapeutic substance, such an antibiotic, on the levels of an entity, such as a light-emitting bacterium, over time.

In another embodiment, the invention includes a noninvasive method for detecting the integration of a transgene in a mammalian subject. The method includes administering to the subject, a vector construct effective to integrate a transgene into mammalian cells. Such constructs are well known in the art. In addition to the elements necessary to integrate effectively, the construct contains a transgene (e.g. a therapeutic gene), and a gene encoding a light-generating protein under the control of a selected activatable promoter. After a period of time in which the construct can achieve integration, the promoter is activated. For example, if an interferon promoter is used, a poly-inosine and -cytosine duplex (poly-IC) can be locally administered (e.g. footpad injection) to stimulate interferon production. The subj ect is then placed within the detection field of a photodetector device and the level of photon scattering is measured, or evaluated. If the level is above background (i.e. if light can be preferentially detected in the "activated" region), the subject is scored as having integrated the transgene. h a related embodiment, the invention includes a noninvasive method for detecting the localization of a promoter-induction event in an animal made transgenic or chimeric for a construct including a gene encoding a light-generating protein under the control of an inducible promoter. Promoter induction events include the administration of a substance which directly activates the promoter, the administration of a substance which stimulates production of an endogenous promoter activator (e.g. stimulation of interferon production by RNA virus infection), the imposition of conditions resulting in the production of an endogenous promoter activator (e.g. heat shock or stress), and the like. The event is triggered, and the animal is imaged as described above.

In yet another embodiment, the invention includes pathogens, such as Salmonella, transformed with a gene expressing a nonlinear-active protein, such as the green fluorescent protein (GFP) or mutants of GFP. In a specific embodiment, the invention includes a method of identifying therapeutic compounds effective to inhibit spread of infection by a pathogen. The method includes administering a conjugate of the pathogen and a nonlinear light-generating moiety to control and experimental animals, treating the experimental animals with a putative therapeutic compound, localizing the light-scattering pathogen in both control and experimental animals by the methods described above, and identifying the compound as therapeutic if the compound is effective to significantly inhibit the spread or replication of the pathogen in the experimental ammals relative to control animals. The conjugates include a nonlinear-active-labeled antibodies, nonlinear-active-labeled particles, nonlinear-active- labeled small molecules, and the like. In a specific embodiment, the invention includes a method of localizing entities conjugated to nonlinear-active-moieties through media of varying opacity. The method includes the use of photodetector device to detect photons transmitted through the medium, integrate the photons over time, and generate an image based on the integrated signal.

In yet another embodiment, the invention includes a method of measuring the concentration of selected substances, such as dissolved oxygen or calcium, at specific sites in an organism. The method includes entities, such as cells, containing a concentration sensor—a nonlinear-active molecule whose ability to generate nonlinear light is dependent on the concentration of the selected substance. The entity containing the light-generating molecule is administered such that it adopts a substantially uniform distribution in the animal or in a specific tissue or organ system (e.g. spleen). The organism is imaged, and the intensity and localization of the nonlinear light is correlated to the concentration and location of the selected substance. Alternatively, the entity contains a second marker, such as a molecule capable of generating light at a wavelength other than the concentration sensor. The second marker is used to normalize for any non-uniformities in the distribution of the entity in the host, and thus permit a more accurate determination of the concentration of the selected substance. In another embodiment, the invention includes a method of identifying therapeutic compounds effective to inhibit the growth and/or the metastatic spread of a tumor. The method includes (i) administering tumor cells labeled with or containing nonlinear light- generating moieties to groups of experimental and control animals, (ii) treating the experimental group with a selected compound, (iii) localizing the tumor cells in animals from both groups by imaging photon emission from the nonlinear light-generating molecules associated with the tumor cells with a photodetector device, and (iv) identifying a compound as therapeutic if the compound is able to significantly inhibit the growth and or metastatic spread of the tumor in the experimental group relative to the control group.

In one embodiment of the invention method, a single type of nonlinear-active species is relied upon for producing the nonlinear optical light emanating from the irradiated body part.

In alternative embodiments, the invention method may additionally comprise the step of administering to the subject one or more supplemental nonlinear optical targeting constructs (e.g., antibodies, or biologically active fragments thereof, having attached nonlinear-active species) that bind to the initial nonlinear-active targeting construct and/or to each other to enhance the nonlinear optical light emanating from the target tissue. For instance, an enhancer-tagged anti-nonlinear optical moiety antibody may be administered to bind to any previously administered nonlinear-optical-moiety-tagged antibody or tumor- avid molecule. The purpose of the supplemental enhancing targeting construct is to increase the intensity of nonlinear optica light from the targeting ligand of the first administered targeting construct and thereby to aid in detection of diseased or abnormal tissue in the body part. Preferably, the targeting construct (e.g., the ligand moiety of the invention targeting construct) is selected to bind to and/or be taken up specifically by the target tissue of interest, for example to an antigen or other surface feature contained on or within a cell that characterizes a disease or abnormal state in the target tissue. As in other diagnostic assays, it is desirable for the targeting construct to bind to or be taken up by the target tissue selectively or to an antigen associated with the disease or abnormal state; however, targeting constructs containing ligand moieties that also bind to or are taken up by healthy tissue or cell structures can be used in the practice of the invention method so long as the concentration of the antigen in the target tissue or the affinity of the targeting construct for the target tissue is sufficiently greater than for healthy tissue in the field of vision so that a fluorescent image representing the target tissue can be clearly visualized as distinct from any fluorescence coming from healthy tissue or structures in the field of vision. For example, colon cancer is often characterized by the presence of carcinoembryonic antigen (CEA), yet this antigen is also associated with certain tissues in healthy individuals. However, the concentration of CEA in cancerous colon tissue is often greater than is found in healthy tissue, so an anti-CEA antibody could be used as a ligand moiety in the practice of the invention. In another example, deoxyglucose is taken up and utilized by healthy tissue to varying degress, yet its metabolism in healthy tissues, except for certain known organs, such as the heart, is substantially lower than in tumor. The known pattern of deoxyglucose consumption in the body can therefore be used to aid in determination of those areas wherein unexpectedly high uptake of deoxyglucose signals the presence of tumor cells.

In one embodiment of the present invention, the disease or abnormal state detected by the invention method can be any type characterized by the presence of a known target tissue for which a specific binding ligand is known. For example, various heart conditions are characterized by production of necrotic or ischemic tissue or production of artherosclerotic tissue for which specific binding ligands are known. As another illustrative example, breast cancer is characterized by the production of cancerous tissue identified by monoclonal antibodies to CA15-3, CA19-9, CEA, or HER2/neu. It is contemplated that the target tissue may be characterized by cells that produce either a surface antigen for which a binding ligand is known, or an intracellular marker (i.e. antigen), since many targeting constructs penetrate the cell membrane. Representative disease states that can be identified using the invention method include such various conditions as different types of tumors, bacterial, fungal and viral infections, and the like. As used herein "abnormal" tissue includes precancerous conditions, necrotic or ischemic tissue, and tissue associated with connective tissue diseases, and auto-immune disorders, and the like. Examples of the types of target tissue suitable for examination using the invention method include cardiac, breast, ovarian, uterine, lung, endothelial, vascular, gastro-intestinal, colorectal, prostatic tissue, endocrine tissue, and the like, as well as combinations of any two or more thereof.

Representative examples of antigens for some common malignancies and the body locations in which they are commonly found are shown in Table I below. Targeting ligands, such as antibodies, for these antigens are known in the art. Representative examples of antigens for some common malignancies and the body locations in which they are commonly found are shown in Table I below. Targeting ligands, such as antibodies, for these antigens are known in the art.

TABLE I

TUMORS WHERE ANTIGEN COMMONLY FOUND

CEA (carcinoembryonic antigen) colon, breast, lung

PSA (prostate specific antigen) prostate cancer

CA-125 ovarian cancer

CA 15-3 breast cancer CA 19-9 breast cancer

HER2/neu breast cancer

.alpha. -feto protein testicular cancer, hepatic cancer

.beta.-HCG (human chorionic gonadotropin) testicular cancer, choriocarcinoma MUC-1 breast cancer

Estrogen receptor breast cancer, uterine cancer

Progesterone receptor breast cancer, uterine cancer

EGFr (epidermal growth factor bladder cancer receptor) h one embodiment of the invention method, the ligand moiety of the targeting construct is a protein or polypeptide, such as an antibody, or biologically active fragment thereof, preferably a monoclonal antibody. The supplemental nonlinear-active targeting construct(s) used in practice of the invention method may also be or comprise polyclonal or monoclonal antibodies tagged with a nonlinear-active label. The term "antibody" as used in this invention includes intact molecules as well as functional fragments thereof, such as Fab, F(ab').sub.2, and Fv that are capable of binding the epitopic determinant. These functional antibody fragments retain some ability to selectively bind with their respective antigen or receptor and are defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;

(2) Fab', the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule;

(3) (Fab').sub.2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab').sub.2 is a dimer of two Fab' fragments held together by two disulfide bonds;

(4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and

(5) Single chain antibody ("SCA"), a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.

Methods of making these fragments are known in the art. (See for example, Harlow & Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York,

1988, incorporated herein by reference). As used in this invention, the term "epitope" means any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Antibody fragments of the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab').sub.2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained therein, which patents are hereby incorporated in their entireties by reference. See also Nisonhoff et al., Arch. Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959; Edelman et al., Methods in Enzymology, Vol. 1, page 422 Academic Press, 1967; and Coligan et al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.

Fv fragments comprise an association of N.sub.H and V.sub.L chains. This association may be noncovalent, as described in Inbar et al., Proc. Νat'l Acad. Sci. USA 69:2659, 1972. Alternatively, the variable chains can be lied by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. See, e.g., Sandhu, supra. Preferably, the Fv fragments comprise V.sub.H and V.sub.L chains connected by a peptide linker. These single-chain antigen binding protehis (sFv) are prepared by constructing a structural gene comprising DΝA sequences encoding the V.sub.H and V.sub.L domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are described, for example, by Whitlow et al., Methods: a Companion to Methods in Enzyology, 2: 97, 1991; Bird et al., Science 242:423-426, 1988; Pack et al., Bio/Technology 11:1271-77, 1993; Sandhu, supra, and Ladner et al., U.S. Pat. No. 4,946,778, which is hereby incorporated by reference in its entirety. Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick et al., Methods: a Companion to Methods in Enzymology, 2: 106, 1991.

Antibodies which bind to a tumor cell can be prepared using an intact polypeptide or biologically functional fragment containing small peptides of interest as the immunizing antigen. The polypeptide or a peptide used to immunize an animal (derived, for example, from translated cDNA or chemical synthesis) can be conjugated to a carrier protein, if desired. Commonly used carriers that are chemically coupled to the peptide include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid, and the like. The coupled peptide is then used to immunize the animal (e.g., a mouse, a rat, or a rabbit). The preparation of such monoclonal antibodies is conventional. See, for example,

Kohler & Milstein, Nature 2:495, 1975; Coligan et al., sections 2.5.1-2.6.7; and Harlow et al., in: Antibodies: a Laboratory Manual, page 726 (Cold Spring Harbor Pub., 1988), which are hereby incorporated by reference. Briefly, monoclonal antibodies can be obtained by injecting mice with a composition comprising an antigen, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B lymphocytes, fusing the B lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures. Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography. See, for example, Coligan et al., sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3; Barnes et al., Purification of hnmunoglobulin G (IgG), in: Methods in Molecular Biology, Vol. 10, pages 79-104 (Humana Press, 1992).

Antibodies of the present invention may also be derived from subhuman primate antibodies. General techniques for raising therapeutically useful antibodies in baboons can be found, for example, in Goldenberg et al, International Patent Publication WO 91/11465

(1991) and Losman et al., 1990, Int. J. Cancer 4:310, which are hereby incorporated by reference. Alternatively, a therapeutically useful antibody may be derived from a "humanized" monoclonal antibody. Humanized monoclonal antibodies are produced by transferring mouse complementarity determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain, and then substituting human residues in the framework regions of the murine counterparts. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions. General techniques for cloning murine immunoglobulin variable domains are described, for example, by Orlandi et al, Proc. Nat'l Acad. Sci. USA 86:3833,1989, which is hereby incorporated in its entirety by reference. Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al., Nature 321 :522, 1986; Riechmann et al., Nature 332:323,

1988; Verhoeyen et al., Science 239:1534, 1988; Carter et al., Proc. Nat'l Acad. Sci. USA 89:4285, 1992; Sandhu, Grit. Rev. Biotech. 12:437, 1992; and Singer et al, J. Immunol. 150:2844, 1993, which are hereby incorporated by reference.

It is also possible to use anti-idiotype technology to produce monoclonal antibodies which mimic an epitope. For example, an anti-idiotypic monoclonal antibody made to a first monoclonal antibody will have a binding domain in the hypervariable region which is the "image" of the epitope bound by the first monoclonal antibody.

In a presently preferred embodiment of the invention method, the ligand moiety in the nonlinear-active targeting construct used in practice of the invention can be selected from among the many biologically compatible tumor-avid compounds that bind with specificity to receptors and/or are preferentially taken up by tumor cells and can be used as the ligand moiety in the invention targeting constructs. Tumor-avid compounds that are preferentially "taken up" by tumor cells may enter the cells through surface or nuclear receptors (e.g., hormone receptors), pores, hydrophilic "windows" in the cell lipid bilayer, and the like.

Illustrative of this class of tumor-avid compounds are somatostatin, somatostatin receptor-binding peptides, deoxyglucose, methionine, and the like. Particularly useful somatostatin receptor-binding peptides are a long-acting, octapeptide analog of somatostatin, known as octreotide (D-phenylalanyl-L-cysteinyl-L-phenylalanyl-D- tryptophyl-L-lysyl-L-threonyl -N-[2-hydroxy- 1 -(hydroxymethyl)propyl]-L-cysteinamide cyclic (2.fwdarw.7)-disulfide), lanreotide, an oral formulation of octreotide, P829, P587, and the like. Somatostatin-binding peptides are disclosed in U.S. Pat. No. 5,871,711, and methods for linking such peptides covalently to a radioisotope through their carboxyl terminal amino acid under reducing conditions are disclosed in U.S. Pat. No. 5,843,401, which are both incorporated herein by reference in their entireties. One of skill in the art can readily adapt such teachings for the preparation of UV sensitive somatostatin receptor- binding peptides by substituting the UV-sensitive fluorescing moieties of this invention in the place of a radioisotope.

Somatostatin and somatostatin receptor-binding peptides are particularly effective for use as the tumor-avid ligand moiety in the targeting construct in the invention diagnostic procedures when the disease state is a neuroendocrine or endocrine tumor. Examples of neuroendocrine tumors that can be diagnosed using the invention method include adenomas (GH-producing and TSH-producing), islet cell tumors, carcinoids, undifferentiated neuroendocrine carcinomas, small cell and non small cell lung cancer, neuroendocrine and/or intermediate cell carcinomas, neuroendocrine tumors of ovary, cervix, endometrium, breast, kidney, larynx, paranasal sinuses, and salivary glands, meningiomas, well differentiated glia-derived tumors, pheochromocytomas, neuroblastomas, ganglioneuro(blasto)mas, paragangliomas, papillary, foUicular and medullary carcinomas in thyroid cells, Merkel cell carcinomas, and melanomas, as well as granulomas and lymphomas. These tumor cells are known to have somatostatin receptors and can be targeted using somatostatin or somatostatin receptor binding peptides as the tumor-avid ligand in the invention fluorescent targeting construct. Vasointestinal peptide (VIP), which is used in VIP receptor scintigraphy (I.

Virgolini, Eur J. Clin. Invest. 27(10):793-800, 1997, is also useful in the invention method for diagnosis of small primary adenocarcinomas, liver metastases and certain endocrine tumors of the gastrointestinal tract.

Another molecule illustrative of the tumor-avid ligands that are preferentially taken up by tumors is deoxyglucose, which is known to be preferentially taken up in a variety of different types of tumors. Illustrative of the types of tumors that can be detected using deoxyglucose as the tumor-avid ligand moiety in the nonlinear-active targeting construct as disclosed herein include melanoma, colorectal and pancreatic tumors, lymphoma (both HD and NHL), head and neck tumors, myeloma, cancers of ovary, cancer, breast, and brain (high grade and pituitary adenomas), sarcomas (grade dependent), hepatoma, testicular cancer, thyroid (grade dependent) small cell lung cancer, bladder and uterine cancer, and the like. Yet other tumor-avid compounds that can be used as the targeting ligand in an invention fluorescing targeting construct are 1-amino-cyclobutane-l-carboxylic acid and L- methionine. L-methionine is an essential amino acid that is necessary for protein synthesis. It is known that malignant cells have altered methionine metabolism and require an external source of methionine.

Methods for obtaining test tumor cells for prescreening to determine the type(s) of tumor-avid compounds that are currently being taken up (e.g., by specific receptors expressed by the tumor cells) are well known in the art. For example, such techniques as fine needle aspirates, scrapings, excisional biopsies, and the like, can in many instances be utilized to obtain test tumor cells relatively non-invasively.

In vitro tests useful for determining the tumor-avid compounds that are being taken up by test tumor cells are numerous and also well known in the art. Such in vitro tests generally involve either sequentially or simultaneously contacting the test cells with a plurality of different tumor-avid compounds. For example, the test cells can be contacted with a panel or library of detectably labeled hormones and/or other known tumor-avid compounds to determine which of the detectably labeled compounds bind to and/or are taken up by the test cells.

Additional examples of biologically compatible tumor-avid compounds that bind with specificity to tumor receptors and/or are preferentially taken up by tumor cells include mammalian hormones, particularly sex hormones, neurotransmitters, and compounds expressed by tumor cells to communicate with each other that are preferentially taken up by tumor cells, such as novel secreted protein constructs arising from chromosomal aberrations, such as transfers or inversions within the clone.

The targeting constructs and supplemental targeting constructs used in practice of the invention method can be administered by any route known to those of skill in the art, such as topically, intraarticularly, intracistemally, intraocularly, intraventricularly, intrathecally, intravenously, intramuscularly, intraperitoneally, intradermally, intratracheally, intracavitarily, and the like, as well as by any combination of any two or more thereof. The most suitable route for administration will vary depending upon the disease state to be treated, or the location of the suspected condition or tumor to be diagnosed. For example, for treatment of inflammatory conditions and various tumors, local administration, including administration by injection directly into the body part to be irradiated by UV light (e.g., intracavitarily) provides the advantage that the targeting construct (e.g., fluorescently tagged antibodies) can be administered in a high concentration without risk of the complications that may accompany systemic administration thereof. The targeting construct is administered in a "diagnostically effective amount." An effective amount is the quantity of a targeting construct necessary to aid in direct visualization of any target tissue located in the body part under investigation in a subject. A "subject" as the term is used herein is contemplated to include any mammal, such as a domesticated pet, farm animal, or zoo animal, but preferably is a human. Amounts effective for diagnostic use will, of course, depend on the size and location of the body part to be investigated, the affinity of the targeting construct for the target tissue, the type of target tissue, as well as the route of administration. Local administration of the targeting construct will typically require a smaller dosage than any mode of systemic administration, although the local concentration of the targeting construct may, in some cases, be higher following local administration than can be achieved with safety upon systemic administration.

Since individual subjects may present a wide variation in severity of symptoms and each targeting construct has its unique diagnostic characteristics, including, affinity of the targeting construct for the target, rate of clearance of the targeting construct by bodily processes, the properties of the fluorophore contained therein, and the like, the skilled practitioner will weigh the factors and vary the dosages accordingly.

The invention composition can also be formulated as a sterile injectable suspension according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1-4, butanediol. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed, including synthetic mono- or diglycerides, fatty acids (including oleic acid), naturally occurring vegetable oils like sesame oil, coconut oil, peanut oil, cottonseed oil, etc., or synthetic fatty vehicles like ethyl oleate, or the like. Buffers, preservatives, antioxidants, and the like, can be incorporated as required, or, alternatively, can comprise the formulation.

The invention targeting constructs can be produced by well known techniques. For example, well known techniques of protein synthesis can be used to obtain proteinaceous components of the targeting construct if the amino acid sequence of the component is known, or the sequence can first be determined by well known methods, if necessary. Some of the ligand genes are now commercially available. An advantage of obtaining commercially available genes is that they have generally been optimized for expression in E. coli. A polynucleotide encoding a protein, peptide or polynuleotide of interest, can be produced using DNA synthesis technology. Methods for obtaining the DNA encoding an unavailable gene and expressing a gene product therefrom are well known and will not be described here in detail.

A targeting construct comprising a proteinaceous ligand moiety, a proteinaceous linker moiety, and a proteinaceous nonlinear-active label (e.g., green fluorescent protein) can also be produced as a fusion protein using well known techniques wherein a host cell is transfected with an expression vector containing expression control sequences operably linked to a nucleic acid sequence coding for the expression of the fusion protein (Molecular Cloning A Laboratory Manual, Sambrook et al., eds., 2nd Ed., Cold Spring Harbor Laboratory, N.Y., 1989).

"Peptide" and/or "polypeptide" means a polymer in which the monomers are amino acid residues which are joined together through amide bonds, alternatively referred to as a polypeptide. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used, the L-isomers being preferred. Additionally, unnatural amino acids such as beta-alanine, phenylglycine, and homoarginine are meant to be included. Commonly encountered amino acids that are not gene-encoded can also be used in the present invention, although preferred amino acids are those that are encodable. For a general review, see, for example, Spatola, A. F., in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, B. Weinstein, ed., Marcel Dekker, New York, p. 267,1983. The term targeting construct, targeting agent, optical contrast agent, marker, tag, label are used interchangeably herein. An advantage of nonlinear optical contrast is that multiple contrast agents, each of distinguishable optical signature in vivo, can be added to provide the power of simultaneous, multiple labeling for different affinities and receptors. A target cancer may be a cell that expresses two or more receptor sites, and double, triple, or multiple contrast agent labeling is achievable using optical contrast agents with unique, differentiable optical characteristics. Another advantage of nonlinear optical contrast agents is that the contrast agent may be administered in a pre-active form, and the production of contrast is then achieved by a bioactivation step in which the contrast agent requires activation through a biological interaction before producing or reducing its native signal. Such interactions include enzymatic processing, conformational changes, receptor binding, gene expression, and the like. For example, a conformational change can be the result of a pH change or of a binding event that swings quenching or resonance-enhancing groups into or out of position, decreasing or increasing the signal in response to binding. Similarly, an enzymatic processing may be an irreversible cleavage that removes quenching moieties from the contrast agent, turning on a strong signal. Last, a bioinactivation step can be used to shut off the contrast in response to a biological event.

The term hormone is used herein to refer to compounds that are expressed within a mammal for action at a remote location and includes such compounds as sex hormones, cell growth hormones, cytokines, endocrine hormones, erytliropoietin, and the like. As is known in the art, a number of tumor types express receptors for hormones, for example, estrogen, progesterone, androgens, such as testosterone, and the like. Such hormones are preferentially taken up by tumor cells, for example, via specific receptors. It is also known in the art that the particular type of receptors expressed by a tumor cell may change over time with the same cell or cell mass, for example, expressing estrogen receptors at one point in time and with the estrogen receptors being substantially replaced with androgen receptors at another point in time.

TERMINOLOGY

The following terms, among others, are used throughout the present specification are intended to have the following general definitions: 1. Complementary: Refers to the topological and chemical compatibility of interacting surfaces between two biological components, such as with a ligand molecule and its receptor (also referred to sometimes in the art as 'molecular recognition'). Thus, the receptor and its ligand can be described as complementary, and, furthermore, the contacts' surface characteristics are complementary to each other. 2. Biological Components (also 'Biological Sample'): This teπn includes any naturally occurring or modified particles or molecules found in biology, or those molecules and particles which are employed in a biological study, including probes and targets. Examples of these include, but are not limited to, cells, tissues, organs, protein, nucleic acids, antibodies, receptors, peptides, small molecules, oligonucleotides, carbohydrates, lipids, liposomes, polynucleotides and others such as drugs, toxins and genetically engineered protein or peptide. 3. Ligand: A ligand is a molecule that is recognized by a particular receptor. Examples of ligands that can be used with the present invention include, but are not restricted to, antagonists or agonists for cell membrane receptors, toxins and venoms, viral epitopes, hormones, hormone receptors, peptides, enzymes, enzyme substrates, cofactors, drugs (e.g. opiates, steroides, etc.), lectins, sugars, oligonucleotides, nucleic acids, oligosaccharides, proteins, and monoclonal antibodies.

4. Receptor: A molecule that has a chemical affinity for a given ligand. Receptors can be naturally occurring or man-made molecules. Also, they can be used in an unaltered state or as aggregates with other biological components. Receptors can be attached, covalently or noncovalently, to a binding partner, either directly or via a specific binding substance. Examples of receptors which can be employed by this invention include, but are not limited to, antibodies, cell membrane receptors, monoclonal antibodies and antisera reactive with specific antigenic determinants (such as on viruses, cells or other materials), drugs, polynucleotides, nucleic acids, peptides, cofactors, lectins, sugars, polysaccharides, cells, cellular membranes and organelles. Receptors are occasionally referred to in the art as anti-ligand. As the term receptors is used herein, no difference in meaning is intended. A "Ligand Receptor Pair" is formed when two macromolecules have combined through molecular recognition to form a complex.

5. Other examples of receptors which can be investigated by this invention include but are not restricted to: a) Microorganism receptors: Determination of ligands which bind to receptors, such as specific transport proteins or enzymes essential to survival of microorganisms, is useful in developing a new class of antibiotics. Of particular value would be antibiotics against opportunistic fungi, protozoa, and those bacteria resistant to the antibiotics in current use. b) Enzymes: For instance, one type of receptor is the binding site of enzymes such as the enzymes responsible for cleaving neurotransmitters; determination of ligands which bind to certain receptors to modulate the action of the enzymes which cleave the different neurotransmitters is useful in the development of drugs which can be used in the treatment of disorders of neurotransmission. c) Antibodies: For instance, the invention can be useful in investigating the ligand- binding site on the antibody molecule which combines with the epitope of an antigen of interest; determining a sequence that mimics an antigenic epitope can lead to the development of vaccines of which the immunogen is based on one or more of such sequences or lead to the development of related diagnostic agents or compounds useful in therapeutic treatments such as for autoimmune diseases (e.g., by blocking the binding of the "self antibodies). d) Nucleic Acids: Sequences of nucleic acids can be synthesized to establish DNA or

RNA binding sequences. e) Catalytic Polypeptides: Polymers, preferably polypeptides, which are capable of promoting a chemical reaction involving the conversion of one or more reactants to one or more products. Such polypeptides generally include a binding site specific for at least one reactant or reaction intermediate and an active functionality proximate to the binding site, which functionality is capable of chemically modifying the bound reactant. Catalytic polypeptides are described in, for example, U.S. Pat. No. 5,215,899, which is incorporated herein by reference for all purposes. f) Hormone receptors: Examples of hormone receptors include, e.g., the receptors for insulin and growth hormone. Determination of the ligands which bind with high affinity to a receptor is useful in the development of, for example, an oral replacement of the daily injections which diabetics must take to relieve the symptoms of diabetes, and in the other case, a replacement for the scarce human growth hormone which can only be obtained from cadavers or by recombinant DNA technology. Other examples are the vasoconstrictive hormone receptors; determination of those ligands which bind to a receptor can lead to the development of drugs to control blood pressure. g) Opiate receptors: Determination of ligands which bind to the opiate receptors in the brain is useful in the development of less-addictive replacements for morphine and related drugs. h) Ion channel proteins or receptors, or cells containing ion channel receptors. 6. Surface-selective: This term refers to a non-linear optical technique such as second harmonic generation or sum/difference frequency generation in which, by symmetry, only a non-centrosymmetric surface (comprising array, substrate, solution, biological components, etc.), is capable of generating non-linear light. 7. Array or Microarray: Refers to a substrate or solid support on which is fabricated one type, or a plurality of types, of biological components in one or a plurality of known locations. This includes, but is not restricted to, two-dimensional microarrays and other patterned samples. Other terms in the art which are often used interchangeably for 'array' include: gene chip, gene array, biochip, DNA chip, protein chip and microarray, the latter being an array with elements of the array (patterned areas with attached probes) whose dimensions are on the order of microns.

8. Label (or 'Targeting Construct' or 'Nonlinear-active Targeting Construct' or 'Tag' or 'Marker', etc.): Refers herein to a nonlinear-active moiety, particle or molecule which can be attached (covalently, immunochemically, non-covalently, etc.) to a molecule, particle or phase (e.g., lipid bilayer) in order to render the latter more nonlinear optical active. The labels can be pre-attached to the molecules or particles and unbound or unreacted labels separated from the labeled entities before a measurement is made. Alternatively, the labels can be left in solution with probes and targets and allowed to adsorb to some particle (e.g., an enhancer) or surface to yield a higher nonlinear active response (i.e., hyperpolarizability or second order susceptibility). These labels, tags, markers, contrast agents or targeting constructs are the nonlinear-active analogues of fas fluorescent tags, markers, labels, contrast agents and targeting constructs found in the biological art where they are also referred to as 'markers' or 'tags'; they are also referred to in the medical imaging art as 'contrast agents' or 'conjugates'. EFISH (Electric-field induced second harmonic generation) or Hyper-Rayleigh scattering can be used to determine if a candidate molecule or particle is nonlinearly active. Electric field induced second harmonic (EFISH) is well known in the field of nonlinear optics. This is a third order nonlinear optical effect, with the polarization source written as: P⁽²⁾(ω₃) = χ⁽²⁾ (-ω₃; ω_lsω₂) : E^ωl E^ω2. The effect can be used to measure the hyperpolarizabilty of molecules in solution by using a dc field to induce alignment in the medium, and allowing SHG to be observed. This is sometimes called the reorientational mechanism. 9. Linker: A molecule which serves to chemically link (usually via covalent bonds) two different objects together. Herein a linker can be used to couple targets to non-linear active particles or moieties, targets to nonlinear-active derivatized particles, surface layers to targets, surface layers to nonlinear-active particle or moieties, etc. A linker can, for example, be a homobifunctional or heterobifunctional cross-linker molecule, a biotin-streptavidin couple wherein the biotin is attached to one of the two objects and the streptavidin to the other, etc. Many linkers are available commercially, for example from Pierce Chemical Inc., Sigma- Aldrich, Fluka, etc. In some art, the term 'tether', 'spacer' or 'cross-linker' is also used with the same meaning. 10. Elements: When used with 'array' or 'microarray', the meaning is a specific location among the plurality of locations on the array surface. Each element is a discrete region of finite area formed on the surface of a solid support or substrate.

11. Nonlinear: Refers herein to those optical techniques capable of transforming the frequency of an incident light beams called the fundamental(s). The nonlinear beams are the higher order frequency beams which result from such a transformation, e.g. second harmonic, etc. In second harmonic, sum frequency or difference frequency generation, the nonlinear beams are generated coherently. In second harmonic generation (SHG), two photons of the fundamental beam are virtually scattered by the interface to produce one photon of the second harmonic. Also referred to herein as nonlinear optical or surface-selective nonlinear (optical) or by various combinations thereof. Nonlinear optical light is any light which results from a nonlinear transformation of the fundamental beam(s). 'Scattered' light referred to herein is nonlinear light generated by a surface-selective nonlinear optical technique.

12. Probe: Refers herein to biological components (eg., cells, proteins, virus, ligand, small molecule, drugs, oligonucleotides, DNA, RNA, cDNA, etc.) which are attached to a surface (e.g., solid substrate, cell surface, liposome surface, etc.), or are cells, lipsomes, particles, beads or other components which comprise a surface e.g. freely suspended in some medium in a sample cell. (In some literature in the art, this term refers to the free components which are tested for binding against the probes). 13. Attached (Attach, Attachment): Refers herein to biological components which are either prepared or engineered in-vitro to be attached to some surface, via covalent or non-covalent means, including for example the use of linker molecules to, for example, a solid substrate, a cell surface, a liposome surface, a gel substrate, etc.; or the probes are found naturally 'attached' to a surface such as in the example of native membrane receptors embedded in cell membranes, tissues, organs (in-vitro or in-vivo). In some instances herein, the word 'attached' or 'attach' refers also to the chemical or physical attachment of a label to a target or decorator. Also referred to herein as 'surface- attached'.

14. Centrosymmetric: A molecule or material phase is centrosymmetric if there exists a point in space (the 'center' or 'inversion center') through which an inversion (x,y,z) -_> (-x,-y,-z) of all atoms is performed that leaves the molecule or material unchanged. A non-centrosymmetric molecule or material lacks this center of inversion. For example, if the molecule is of uniform composition and spherical or cubic in shape, it is centrosymmetric. Centrosymmetric molecules or materials have no nonlinear susceptibility or hyperpolarizability, necessary for second harmonic, sum frequency and difference frequency generation. 15. Nucleic Acid Analog: A non-natural nucleic acid which can function as a natural nucleic acid in some way. For example, a Peptide Nucleic Acid (PNA) is a non-natural nucleic acid because it has a peptide-like backbone rather than the phosphate background of natural nucleic acids. The PNAs can hybridize to natural nucleic acids via base-pair interactions. 16. Another example of a Nucleic acid analog can be one in which the base pairs are non- natural in some way.

17. Decorator: Refers herein to a nonlinear active molecule or particle (possesses a hyperpolarizability) which can be bound to targets, probes or target-probe complexes in order to allow the detection and discrimination between them. A decorator should not appreciably alter or participate in the target-probe reaction itself. The decorator can be dissolved or suspended in the solution or aqueous phase containing the target component. A decorator is distinguished from an SH-active label (J.S. Salafsky, co- pending application 'SH-labels...') for its specific binding affinity for targets, probes, or the target-probe complex. In the art (J.S. Salafsky K.B. Eisenthal, co-pending application 'SHG labels...'), an SHG-label is attached to a biological component - via specific chemical bonds or non-specific (e.g., electrostatic) means - and then used to follow that component to an interface. A decorator can be used to detect probe-target complexes by its specific binding affinity (in some art, 'molecular recognition') to the targets, probes or the target-probe complexes.

18. Binding Affinity or Affinity: The specific physico-chemical interactions between binding partners, such as a probe and target, which lead to a binding complex (affinity) between them. The binding reaction is characterized by an equilibrium constant which is a measure of the energetic strength of binding between the partners. Specificity in a binding reaction implies that probe-target binding only occurs appreciably with specific binding partners - not any at random. For example, the protein immunoglobulin G (IgG) has a specific binding affinity for protein G and less or none for other proteins. In some art, the term 'molecular recognition' is used to describe the binding affinity between components.

19. Electrically Charged or Electric Charge: Defined herein as net electric charge on a particle or molecule, which confers a mobility (velocity) of said particle or molecule in an electric field. The net charge could be part of a molecular moiety such as phosphate group on nucleic acid backbones, side-chains of amino acid residues in proteins, lipid head groups in membrane lipids or cellular membranes, etc. The charge can be positive or negative and would determine the direction of mobility of the particle or molecule if said particle or molecule is placed in an electric field of a given orientation (direction of positive to negative electric potential). The charge can be non-integer multiples of the fundamental unit of charge (q « 1.6 x 10^"19 C) or a fraction of the fundamental unit of charge - so-called 'partial charges', well known to those skilled in the art.

20. Dipolar: Defined herein as possessing an electric dipole or 'dipole moment' on a particle or molecule, which takes the standard definition known to one skilled in the art: the sum of all vectors μ = Q-R where Q is the amount of charge (positive or negative) at a particular spatial location (x,y,z in Cartesian coordinates) in the particle or molecule and R is the vector which points from an origin of reference (x,y,z) to the net charge Q. If the sum of these vectors results in a vector with a non-zero trace (sum of x,y,z components of the resultant vector), the particle or molecule possesses a dipole moment and is electrically dipolar. 21. Electrically Neutral: Defined herein as zero net (sum of positive and negative) electric charge on a particle or molecule, which would result in no appreciable mobility (velocity) of said particle or molecule in an electric field. 22. Hyperpolarizability or Nonlinear Susceptibility: The properties of a molecule, particle, interface or phase which allow for generation of the nonlinear light. Typical equations describing the nonlinear interaction for second harmonic generation are: α⁽²⁾(2ω) = β:E(ω)-E(ω) or P⁽²⁾(2ω) = χ⁽²⁾:E(ω)E(ω) where α and P are, respectively, the induced molecular and macroscopic dipoles oscillating at frequency 2ω, β and χ⁽²⁾ are, respectively, the hyperpolarizability and second-harmonic (nonlinear) susceptibility tensors, and E(ω) is the electric field component of the incident radiation oscillating at frequency ω. The macroscopic nonlinear susceptibility χ⁽²⁾ is related by an orientational average of the microscopic β hyperpolarizability. For sum or difference frequency generation, the driving electric fields (fundamentals) oscillate at different frequencies

(i.e., ωi and ω₂) and the nonlinear radiation oscillates at the sum or difference frequency (G>_I ± ω₂). The terms hyperpolarizability, second-order nonlinear polarizability and nonlinear susceptibility are sometimes used interchangeably, although the latter term generally refers to the macroscopic nonlinear-activity of a material or chemical phase or interface. The terms 'nonlinear active' or 'nonlinearly active' used herein also refer to the general property of the ability of molecules, particles, an interface or a phase, to generate nonlinear optical radiation when driven by incident radiation beam or beams.

23. Polarization: The net dipole per unit volume (or area) in a region of space. The polarization can be time-dependent or stationary. Polarization is defined as: / μ(R) dR where an integration of the net dipole is made over all volume elements in space dR near an interface.

24. Radiation: Refers herein to electromagnetic radiation or light, including the fundamental beams used to generate the nonlinear optical effect, or the nonlinear optical beams which are generated by the fundamental. Also referred to herein as 'waves', 'signal' or 'nonlinear signal', 'beams', 'light'.

25. Near-field techniques: Those techniques known in the art to be capable of measuring or imaging optical radiation on a surface or substrate with a lateral resolution at or smaller than the diffraction-limited distance. Examples of near-field techniques (or near-field imaging) include NSOM (near-field scanning optical microscopy), whereby optical radiation (from fluorescence, second harmonic generation, etc.) is collected at a point very near the surface. 26. Detecting, Detection: When referring herein to nonlinear optical methods, refers to those techniques by which the properties of surface-selective nonlinear optical radiation can be used to detect, measure or correlate properties of probe-target interactions, or effects of the interactions, with properties of the nonlinear optical light (e.g., intensity, wavelength (and therefore frequency), polarization or other property common to electromagnetic radiation).

27. Interface: For the purpose of this invention, the interface can be defined as a region which generates a nonlinear optical signal or the region near a surface in which there are nonlinear-active labeled targets possessing a net orientation. An interface can also be composed of two surfaces, a surface in contact with a different medium (e.g., a glass surface in contact with an aqueous solution, a cell surface in contact with a buffer), the region near the contact between two media of different physical or chemical properties, etc.

28. Conjugated, Coupled: Refers herein to the state in which one particle, moiety or molecule is chemically bonded, covalently or non-covalently linked or by some means attached to a second particle moiety, molecule, surface or substrate. These means of attachment can be via electrostatic forces, covalent bonds, non-covalent bonds, physisorption, chemisorption, hydrogen bonds, van der Waal's forces or any other force which holds the probes with a binding energy to the substrate (a corallery to this definition is that some force is required to separate the probes held by the substrate from the substrate).

29. Reactions: Refers herein to chemical, physical or biological reactions including, but not limited to, the following: probes, targets, inhibitors, small molecules, drugs, antagonists, antibodies, etc. The term 'effects of reactions' or 'effects of said reactions' refers herein to physical or chemical effects of the probe-target reactions: for example, the probe-target reactions can comprise a ligand-receptor binding reaction which leads, in turn, to an ion channel opening and a change in the surface charge density of a cell, the latter being then detected by the nonlinear optical technique. The effects of the probe-target reactions, or the probe-target reactions themselves, might be referred in some art as a 'second messenger' reaction. Also referred to herein as 'interactions'.

30. Surface layer: Refers herein to a chemical layer which functionally derivatizes the surface of a solid support. For instance, the surface chemical groups can be changed by the derivatization layer according to the particular chemical functionality of the derivatizing agent. In the case of solid objects used as 'scaffolds' for creating power nonlinear-active labels (see below), the solid surface can be derivatized to produce a different chemical functionality which can be presented to nonlinear active moieties or particles, or to targets. For instance, a silica bead with negatively charged silanol groups on its surface can be converted to an amine-reactive, amine-containing, etc. surface via organosilane reagents.

31. Delivery, Illumination, Collection: hi the context of manipulation of optical radiation (e.g., light beams), delivery and illumination refer herein to the guiding of the fundamental beam to the interface or regions of interest at an interface; collection refers to the optical collection of the nonlinear light produced at the interface (e.g., second harmonic light).

32. Inhibitor, inhibiting: Defined herein as moieties, molecules, compounds or particles which bind to probes in competition with targets; the probe-target interactions are decreased or prevented in the presence of an inhibitor compound, molecule or particle.

Blocking agents refers herein to those compounds, molecules, moieties or particles which prevent probe-target interactions (e.g., binding reactions between probes and targets).

33. Agonist: Defined herein as moieties, molecules, compounds or particles which activate an intracellular response when they bind to a receptor.

34. Antagonist: Defined herein as moieties, molecules, compounds or particles which competitively bind to a receptor on a cell surface at the same site as agonists, but which do not activate the intracellular response initiatied by the active form of the receptor (e.g., activated by agonist binding), and can thereby inhibit the intracellular responses of agonists or partial agonists. Antagonists do not diminish the baseline intracellular response in the absence of an agonist or partial agaonist.

35. Partial Agonist: Defined herein as moieties, molecules, compounds or particles which activate the intracellular response when they bind to a receptor on the cell surface to a lesser degree or extent than do agonists. 36. Interactions: Defined herein as some physical or chemical reaction or interaction between components in a sample. For example, the interactions can be physico- chemical binding reactions between a probe and a target, dipole-dipole attraction or repulsion between two molecules, van der Waals interactions between two atomic or molecular species, a chemical affinity interaction, a covalent bond between molecules, a non-covalent bond between molecules, an electrostatic interaction (repulsive or attractive), a hydrogen bond and others. 37. Effects: Defined herein as the measurable properties of probe-target interactions or the consequences of the interactions (e.g., secondary reactions, ion channel opening or closing, etc.). These include, the following properties, for example: i) the intensity of the nonlinear or fundamental light. ii) the wavelength or spectrum of the nonlinear or fundamental light. iii)position of incidence of the fundamental light on the surface or substrate (e.g., for imaging). iv)the time-course of either i), ii) or iii). v) one or more combinations of i), ii), iii) and iv).

38. Time-course: Refers herein as the change in time of some measurable experimental such as light intensity or wavelength of light. Also referred to as 'kinetics' of some probe-target interaction, or probe-target-other component interaction for example.

39. Well-defined: In the context of 'well-defined direction', refers herein to the deterministic scattering of light (fundamental or nonlinear beams) from a substrate. By contrast, for example, fluorescence emission is emitted at somewhat random directions. 40. Sample: Contains the probes, targets or other molecules, particles or moieties under study by the invention. The sample contains at least one interface capable of generating the nonlinear optical light, with said interface comprised of at least one surface containing attached probes. Examples of components of samples include prisms, wells, microfluidics, substrates, buffer with targets, drugs in buffers, surfaces with attached probes. The terms 'substrate' and 'surface' are often used interchangeably herein. In some cases, the term 'support' can be construed to mean 'surface'.

41. Modulator, Modulates: This term refers herein to any substance, moiety, molecule, biological component or compound which influences the kinetic or equilibrium properties of probe-target interactions (e.g., binding reaction). Modulators may change the rate of probe-target binding, the equilibrium constant of probe-target binding or, in general, enhance or reduce probe-target interactions. Examples of modulators are the following: inhibitors, drags, small molecules, agonists and antagonists.

42. Nucleic Acid Analog: A non-natural nucleic acid which can function as a natural nucleic acid in some way. For example, a Peptide Nucleic Acid (PNA) is a non-natural nucleic acid because it has a peptide-like backbone rather than the phosphate background of natural nucleic acids. The PNAs can hybridize to natural nucleic acids via base-pair interactions. Another example of a Nucleic acid analog can be one in which the base pairs are non-natural in some way.

43. Indicator: Refers herein to a nonlinear active molecule or particle (possesses a hype olarizability) whose nonlinear optical properties or orientation near a surface or interface is modulated as the electric charge polarization, charge density or potential of the surface is modulated, hi one aspect of the invention, the charge or potential of an interface is modulated by the binding of a target to probes immobilized on the surface, hi another aspect, the surface electric potential of a cell is changed by a change in the ion channel properties - an opening, closing, increase or decrease in ionic permeability in response to target (ligand) binding, for instance. In another aspect of the invention, an indicator serves as a marker for imaging purposes, e.g., to image cells or tissues. An indicator does not appreciably alter or participate in the target-probe reaction itself. The indicator can be dissolved or suspended in the liquid, medium, solution or aqueous phase containing the target component. An indicator as defined herein does not translocate into the lipid bilayer of vesicles or cells. An indicator must possess freedom of movement to respond to changes in surface electric charge density or potential. Measuring the nonlinear optical response of a glass-solvent or glass-water interface, in the presence of dissolved or suspended indicators in the water or solvent, would be one means of assaying whether a candidate molecule or indicator would function as an indicator: because glass carries a net negative charge, if the intensity of the nonlinear optical radiation generated by the interface in the presence of the molecule is greater than the background without it, the molecule could function as an indicator. Another means of assaying for a candidate molecule's ability as an indicator is by measuring the intensity of nonlinear optical radiation generated by a semiconductor-liquid interface as a function of applied voltage (and hence surface electric charge density) between the semiconductor and the bulk of the liquid. Yet another means is to measure the hyper- rayleigh scattering (HRS) from a solution or suspension of the indicator candidates; if HRS is generated and the candidate itself is charged or dipolar, it may serve well as an indicator.

44. Binding Affinity or Affinity or Chemical Affinity: The specific physico-chemical interactions between binding partners, such as a probe and target, which lead to a binding complex (affinity) between them. The binding reaction is characterized by an equilibrium constant which is a measure of the energetic strength of binding between the partners. Specificity in a binding reaction implies that probe-target binding only occurs appreciably with specific binding partners - not any at random. For example, the protein Immunoglobulin G (IgG) has a specific binding affinity for protein G and less or none for other proteins. In some art, the term 'molecular recognition' is used to describe the binding affinity between components.

45. Electrically Charged or Electric Charge: Defined herein as net electric charge on a particle or molecule, which confers a mobility (velocity) of said particle or molecule in an electric field. The net charge could be part of a molecular moiety such as phosphate group on nucleic acid backbones, side-chains of amino acid residues in proteins, lipid head groups in membrane lipids or cellular membranes, etc. The charge can be positive or negative and would determine the direction of mobility of the particle or molecule if said particle or molecule is placed in an electric field of a given orientation (direction of positive to negative electric potential). The charge can be non-integer multiples of the fundamental unit of charge (q « 1.6 x 10^"19 C) or a fraction of the fundamental unit of charge - so-called 'partial charges', well known to those skilled in the art.

46. Enhancer (also 'Resonance Enhancers'): A moiety, molecule or particle which can enhance (increase) the cross-section of a nonlinear-active moiety, molecule or particle when placed near to it (e.g., increase the intensity of second harmonic radiation generated). Examples of the enhancement effect referred to in the art include

'resonance enhancement' and 'surface enhancement'. Enhancement of the nonlinear- active cross-section moiety, molecule or particle can occur via a resonance with an electronic transition or plasmon resonance of the enhancer. Candidates for an enhancer can be tested, for example, by measuring the SHG intensity of a nonlinear-active species in the absence and presence of the candidate enhancer (the enhancer can be attached to the nonlinear active label, through a linker if necessary; or the enhancer can be brought into proximity to the label, e.g. by virtue of the probe-target reaction). The enhancer's effect on a nonlinear active label can be made at an interface (e.g., air-water or solid- liquid) or in bulk phase under the application of an electric field (EFISH). Examples of enhancers include metal particles (Au, Ag for example), especially non- centrosymmetric particles.

47. Species: A molecule, moiety or particle. 48. Addition: When used herein to refer to the addition of an enhancer on to, or near to, a molecule or surface, this means the addition of the enhancer to the sample (e.g., sample solution, sample cell, etc.) with an end result that the enhancer is coupled to a the molecule or surface (e.g., covalently, electrostatically, non-covalently, etc.: "on to") or is not coupled directly to the molecule or surface, but rather is near to the molecule or surface (e.g., enhancer adsorption on to a cell surface with a nonlinear-active labeled virus binding to surface; the binding process brings the virus into proximity with the cell surface and thus the adsorbed enhancers, causing the enhancer to increase the nonlinear response of the label on the virus).

49. Array: Defined herein as two or more distinct regions of a surface, substrate or support containing probes localized to the surface, substrate or support and wherein the biological or chemical identity of the probes in said regions is known or pre-defined. Biological identity refers to a probe whose biological function is at least partially known but whose chemical identity is unknown or not fully known. Chemical identity refers to the precise chemical identity of a given probe, i.e. a specific amino-acid or oligonucleotide sequence. Probes localized on a sample in this way are referred to as being patterned or in an array format. The physical dimensions of the probe- containing regions can vary. For example, the "spots" containing probes can be circular, monomolecular or multi-molecular, molecularly ordered or disordered, and range in size from aangstroms to centimeters or more. When the dimensions of each element in the array (each probe-containing region) are on micron scale, the array can be referred to, as is found often in the prior art, as a "microarray".

50. Identity: In the context of some species, "identity" refers herein to chemical or biological identity. Biological identity refers to a probe whose biological function is at least partially known but whose chemical identity is unknown or not fully known. Chemical identity refers to the precise chemical identity of a given probe, i.e. a specific amino-acid or oligonucleotide sequence. 51. Surface, substrate or support: Refers herein to a surface that comprises any biological, chemical or physical interface between two media. For example, a surface, substrate or support can be solid, liquid, gel-like, lattice-like, web-like, a biological cell or liposome, etc. 52. Specific binding ('specific reactions', 'specific', 'specific interactions': Refers herein to probe-target interactions which are complementary and specific (e.g., involves chemical discrimination), ie. a "molecular recognition" reaction as it is referred to often in the art. A specific reaction results in a specific physical conformation or set of conformations of the probe-target complex. The term 'non-specific' implies an interaction not having specificity. An example of a non-specific reaction is an adsorption process of a protein or oligonucleotide to a glass surface. The adsorbed protein or oligonucleotide will generally display a random orientation with respect to the surface plane and this result can be termed a 'random' adsorption process.

53. Oligonucleotide is a nucleic acid sequence composed of two or more nucleotides. An oligonucleotide is optionally derived from natural sources, but is often synthesized chemically. It is of any size. An "oligonucleotide analogue" refers to a polymer with two or more monomeric subunits, wherein the subunits have some structural features in common with a naturally occurring oligonucleotide which allow it to hybridize with a naturally occurring oligonucleotide in solution. For instance, structural groups are optionally added to the ribose or base of a nucleoside for incorporation into an oligonucleotide, such as a methyl or allyl group at the 2'-O position on the ribose, or a fluoro group which substitutes for the 2'-O group, or a bromo group on the ribonucleoside base. The phosphodiester linkage, or "sugar-phosphate backbone" of the oligonucleotide analogue is substituted or modified, for instance with methyl phosphonates or O-methyl phosphates. Another example of an oligonucleotide analogue for purposes of this disclosure includes "peptide nucleic acids" in which native or modified nucleic acid bases are attached to a polyamide backbone. Oligonucleotide analogues optionally comprise a mixture of naturally occurring nucleotides and nucleotide analogues. However, an oligonucleotide which is made entirely of naturally occurring nucleotides (i.e., those comprising DNA or RNA), with the exception of a protecting group on the end of the oligonucleotide, such as a protecting group used during standard nucleic acid synthesis is not considered an oligonucleotide analogue for purposes of this invention. 54. Non-random orientation: A necessary condition of for generation of the surface- selective nonlinear optical signal at an interface comprising probes and targets. Non- random orientation of probes with respect to the surface plane to which they are attached or localized leads to non-random orientation of targets when these targets bind to the probes. When the present invention is used with labels or decorators, this non- random target orientation produces a non-random label or decorator orientation and this leads directly to an increase in surface-selective nonlinear optical signal (e.g., intensity). Non-specific of the targets with attached or localized probes (e.g., non-specific binding to probes) or of targets to regions on the surface where no probe is present (e.g., non- specific binding to substrate or solid support) will lead to zero or a much lower surface- selective nonlinear optical signal due to destructive interference as is well known in the art. When the present invention is used with indicators, a non-random probe orientation also leads to an increase in the surface-selective nonlinear optical signal since the surface charge density close to the surface plane will be larger than if the probes are randomly oriented which results in a lower surface electric charge density.

55. Reaction, Reacted: As defined in the art: chemical reactions that can occur in a given phase, in the presence or absence of an interfacial region.

56. Modifying: Any physiochemical interaction or attachment which occurs between a surface, substrate or support to be imaged with any one or more of the group consisting of a targeting construct, label, decorator, indicator and enhancer. Modifying may occur by any means, including covalent attachment, other physicochemical interaction including van der Waals forces, hydrophobic or hydrophilic interactions, ionic bonding, etc. and may involve a chemical reaction or a simple addition of a component in solution/suspension to effect the modification. As used in the present invention, the term "modifying" includes the physical-chemical attachment or addition of any species

(e.g., label, decorator, enhancer, indicator, etc.) to a sample or any part of said sample such as a probes, targets, interfacial regions, surface, medium containing dissolved or suspended targets, etc. Physical-chemical attachment can comprise a covalent, noncovalent, van der waals, electrostatic, chemisorption, physisorption or ionic interaction, or any other interaction

57. Detector: h the context of measuring or detecting light, any device for measuring a physical property (e.g., intensity) of light such as a photodiode, photomultiplier, CCD array, etc. A monochromator is a detector of frequency of light. 58. Biopsy: A procedure used to remove cells or tissues in order to look at them under a microscope to check for signs of disease. When an entire tumor or lesion is removed, the procedure is called an excisional biopsy. When only a sample of tissue is removed, the procedure is called an incisional biopsy or core biopsy. When a sample of tissue or fluid is removed with a needle, the procedure is called a needle biopsy or fine-needle aspiration.

59. Optical Biopsy: A procedure to image tissues or organs in-situ, usually in a living animal, using a catheter-endoscope, for example.

60. Nonlinear Optical Contrast Agent: Refers herein to a nonlinear optical label, decorator, indicator or enhancer, or some combination thereof, used, for example, to improve the signal to noise of an imaging technique in animals or to identify specific receptors or other probes that may be indicative of a disease state or the onset of a disease.

61. Targeting Construct (Target): A conjugate comprised of a nonlinear-active species or an enhancer conjugated to a species that has a specific interaction with a target (a target being a molecule, tissue, cell, organ or other biological entity in the sample to be imaged). A targeting construct can be an exogeneous or endogeneous species (e.g., a nonlinear-active protein such as GFP fused to a targeting sequence or gene using well known methods in the art).

62. Imaging, imaged: The technique of detecting nonlinear optical-light generating species under irradiation by a fundamental beam in a biological sample using a surface-selective nonlinear optical technique. 'Detecting' comprises using a photodetector for imaging using well known methods in the art or detecting the nonlinear optical light generated by irradiation by a fundamental beam by direct viewing.

63. Nonlinear activity, Nonlinear Active: A species which has a hyperpolarizability or ability to generate nonlinear optical light when illuminated with fundamental light.

Many nonlinear-active species are known in the art and include the following and their derivatives:

Oxazole or oxadizole molecules

5-aryl-2-(4-pyridyl)oxazole 2-aryl-5-(4-pyridyl)oxazole

2-(4-pyridyl)cycloalkano[d]oxazoles Merocyanines Stilbenes Indodicarbocyanines

Hemicyanines

Stilbazims

Azo dyes Cyanines

Stryryl-based dyes

Methylene blue

Diaminobenzene compounds

Polyenes Diazostilbenes

Tricyanovinyl aniline

Tricyanovinyl azo

Melamines

Phenothiazine-stilbazole Polyimides

Sulphonyl-substituted azobenzenes

Indandione-l,3-pyidinium betaine

Fluoresceins

Benzooxazoles Perylenes

Polymethacrylates

Oxonols

Thiophenes

Bithiophenes In evaluating whether a species may be nonlinear-active, the following characteristics can indicate the potential for nonlinear activity: a large difference dipole moment (difference in dipole moment between the ground and excited states of the molecule), a large Stokes shift in fluorescence, an aromatic or conjugated bonding character. In further evaluating such a species, an experimenter can use a simple technique known to those skilled in the art to confirm the nonlinear activity using, for example, detection of SHG from an air-water interface or from EFISH in the absence and presence of the species in question in a medium. Once a suitable nonlinear active species has been selected for the experiment at hand, the species can be conjugated, if desired, to a species with specificity to a biological target to produce a targeting construct used in the surface- selective nonlinear optical detection or imaging technique.

A preferred embodiment of the present invention provides a method for in vivo identification of tissue associated with a disease state in a subject in need thereof using a nonlinear active marker. The invention method comprises administering to the subject a diagnostically effective amount of a nonlinear-active targeting construct comprising a tumor-avid moiety (and optionally a decorator, label or enhancer) so as to allow the nonlinear-active targeting construct to bind to and/or be taken up by the target tissue, irradiating a body part of the subject suspected of containing the target tissue with fundamental light and directly viewing second harmonic radiation emanating from the nonlinear-active targeting construct bound to or taken up by the targeting tissue so as to determine the location and/or surface area of the target tissue in the body part. In an alternative embodiment, a fiber optic line can be used to deliver the fundamental light and collect the nonlinear optical light according to procedures well known in the art for fiber-optic-based imaging. hi an alternative embodiment, microscopy can be used to image the biological samples according to procedures well known to one skilled in the art of microscopy and surface-selective nonlinear optical techniques . hi an alternative embodiment, the putative disease site is a natural body cavity or surgically produced interior site, and an endoscopic device can be optionally used to deliver the fundamental light to the site, to receive nonlinear radiation (e.g., second harmonic radiation) emanating from the site, and to aid in formation of a second harmonic intensity (and/or wavelength) image of the second harmonic radiation from the diseased tissue. For example, a lens in the endoscopic device can be used to focus the detected radiation as an aid in formation of the image. Alternatively, the fundamental light may be directed into a body cavity or surgical opening by any convenient means and the second harmonic image so produced can be directly visualized by the observer without aid from an endoscope. In an alternative embodiment, the invention method may additionally comprise the step of administering to the subject one or more supplemental resonance-enhancing targeting constructs (e.g., antibodies, or biologically active fragments thereof, having attached resonance-enhancers) that bind to the initial nonlinear-active targeting construct and/or to the target tissue to enahance the nonlinear optical signal emanating from the target tissue. For instance, a metallic Au particle-tagged antibody may be administered to bind to any previously administered nonlinear-active label-tagged antibody or tumor-avid molecule. hi another embodiment according to the present invention, the invention diagnostic method comprises prescreening of target tumor cells to determine which receptors are currently being expressed by the target cells. In this embodiment, the invention diagnostic method comprises contacting sample(s) of tumor cells obtained from a subject in vitro with a plurality of detectably labeled tumor-avid compounds, and determining which of the tumor-avid compounds bind to or are taken up by the sample cells. The invention diagnostic method further comprises administering to the subject a diagnostically effective amount of one or more biologically compatible targeting constructs, each comprising as ligand moiety at least one of the tumor-avid compounds determined to bind to and/or be taken up by the tumor cells so as to allow the targeting construct to bind to and/or be taken up selectively in vivo by tumor tissue, irradiating an in vivo body part of the subject suspected of containing the tumor tissue with fundamental light having at least one wavelength in the nonlinear- active response spectrum of the targeting construct. Of course, if the tests determine that the tumor cells are concurrently taking up more than one tumor-avid compound in substantial proportion (e.g., both estrogen and progesterone), the more than one tumor avid compound so determined can be used as the tumor-avid ligand moieties in the targeting constructs in the invention diagnostic method.

In one embodiment of the present invention, the nonlinear-active species (and optionally an enhancer) linked to the tumor-avid compound used as the ligand moiety in the targeting construct by any method presently known in the art for attaching two moieties, so long as the attachment of the linker moiety to the ligand moiety does not substantially impede binding of the targeting construct to the target tissue and/or uptake by the tumor cells, for example, to a receptor on a cell. Those of skill in the art will know how to select a ligand/linker pair that meets this requirement. For example, with regard to octreotide, it has been shown that coupling of a linker to Tyr3 or Phel of octreotide does not prevent the internalization of octreotide after binding to the somatostatin receptor (L. J. Hofland et al., Proc. Assoc. Am. Physicians 111:63-9, 1999). It is also known that 1-amino-cyclobutane-l- carboxylic acid can be tagged at the 3 carbon of the ring.

The length of the optional linker moiety is chosen to optimize the kinetics and specificity of ligand binding, including any conformational changes induced by binding of the ligand moiety to a target, such as an antigen or receptor. The linker moiety should be long enough and flexible enough to allow the ligand moiety and the target to freely interact and not so short as to cause steric hindrance between the proteinaceous ligand moiety and the target. In one embodiment, the linker moiety is a heterobifunctional cleavable cross-linker, such as N-succinimidyl(4-iodoacetyl)-aminobenzoate; sulfosuccinimidyl(4-iodoacetyl)- aminobenzoate; 4-succinmidyl-oxycarbonyl-.alpha.-(2-pyridyldithio) toluene; sulfosuccinimidyl-6-[.alpha.-methyl-.alpha.-(pyridyldithiol)-toluamido] hexanoate; N- succinimidyl-3-(-2-pyridyldithio)-proprionate; succinimidyl-6-[3(-(-2-pyridyldithio)- proprionamido] hexanoate; sulfosuccinimidyl-6-[3(-(-2-pyridyldithio)-propionamido] hexanoate; 3-(2-pyridyldithio)-propionyl hydrazide, Ellman's reagent, dichlorotriazinic acid, S-(2-thiopyridyl)-L-cysteine, and the like. Further bifunctional linking compounds are disclosed in U.S. Pat. Nos. 5,349,066, 5,618,528, 4,569,789, 4,952,394, and 5,137,877, each of which is incorporated herein by reference in its entirety.

These chemical linkers can be attached to purified ligands using numerous protocols known in the art, such as those described in Pierce Chemicals "Solutions, Cross-linking of Proteins: Basic Concepts and Strategies," Seminar #12, Rockford, 111. In another embodiment presently preferred, the linker moiety is a peptide having from about 2 to about 60 amino acid residues, for example from about 5 to about 40, or from about 10 to about 30 amino acid residues. This alternative is particularly advantageous when the ligand moiety is proteinaceous. For example, the linker moiety can be a flexible spacer amino acid sequence, such as those known in single-chain antibody research. Examples of such known linker moieties include GGGGS (SEQ ID NO: 1), (GGGGS).sub.n (SEQ ID NO:l), GKSSGSGSESKS (SEQ ID NO:3), GSTSGSGKSSEGKG (SEQ ID NO:4), GSTSGSGKSSEGSGSTKG (SEQ ID NO:5), GSTSGSGKSSEGKG (SEQ ID NO:6), GSTSGSGKPGSGEGSTKG (SEQ ID NO:7), EGKSSGSGSESKEF (SEQ ID NO:8), SRSSG (SEQ ID NO:9), SGSSC (SEQ ID NO: 10), and the like. A Diphtheria toxin trypsin sensitive linker having the sequence

AMGRSGGGCAGNRVGSSLSCGGLNLQAM (SEQ ID NO:l 1) is also useful. Alternatively, the peptide linker moiety can be VM or AM, or have the structure described by the formula: AM(G.sub.2 to 4 S).sub.x QAM wherein Q is selected from any amino acid and X is an integer from 1 to 11. Additional linking moieties are described, for example, in Huston et al., PNAS 85:5879-5883, 1988; Whitlow, M., et al., Protein Engineering 6:989- 995, 1993; Newton et al., Biochemistry 35:545-553, 1996; A. J. Cumber et al., Bioconj. Chem. 3:397-401, 1992; Ladumer et al., J. Mol. Biol. 273:330-337, 1997; and U.S. Pat. No. 4,894,443, the latter of which is incorporated herein by reference in its entirety.

The mode of delivering or generating the nonlinear optical light (e.g., SHG) can be based on one or more of the following means: TIR (Total internal reflection), Fiber optics (with or without attached beads), Transmission (fundamental passes through the sample), Reflection (fundamental is reflected from the sample), scanning imaging (allows one to scan a sample), confocal imaging or scanning, resonance cavity for power build-up, multiple-pass set-up.

Measured information can take the form of a vector which can include one or more of the following parameters: {intensity of light (typically converted to a photo voltage by a PMT or photodiode), wavelength of light (determined with a monochromator and/or filters), time, substrate position (for array samples, for instance, where different sub-samples are encoded as function of substrate location and the fundamental is directed to various (x,y) locations}. Two general configurations of the apparatus are: image scanning (imaging of a substrate - intensity, wavelength, etc. as a function of x,y coordinate) and spectroscopic (measurement of the intensity, wavelength, etc. for some planar surface or for a suspension of cells, liposomes or other particles).

The fundamental beam can be delivered to the sample in a variety of ways. Figs. 7A-12C are schematics of various modes of delivering the fundamental and generating second harmonic beams. It is understood that in sum- or difference-frequency configurations, the fundamental beams will be comprised of two or more beams, and will generate, at the interfaces, the difference or sum frequency beams. For the purposes of illustration, only the second harmonic generation case is described in detail herein. Furthermore, it shall be understood that the sample cell 3 in all cases can be mounted on a translation stage (1-, 2-, or 3-dimensional degrees of freedom) for selecting precise locations of the interfacial interaction volume. The sample cell in all cases can be fitted with flow ports and tubes which can serve to introduce (or flush out) components such as molecules, particles, cells, etc.

TRANSMISSION

Fig. 8 A is a schematic of a configuration relying on transmission of the fundamental and second harmonic beams. The fundamental 320 (ω) passes through the sample cell 330 and interacts within a volume element (denoted by the circle) in which are contained one or more interfaces capable of generating the second harmonic beam 325 (2ω). The fundamental and second harmonic beams are substantially co-linear as denoted by beam 325. The sample cell can contain suspended beads, particles, liposomes, biological cells, etc. in some medium, providing interfacial area capable of generating second harmonics in response to the fundamental beam. As shown, the second harmonic is detected co-linearly with the fundamental direction, but could alternatively be detected off-angle from the fundamental, for instance at 90° to the fundamental beam.

Fig. 8B is a schematic of another configuration relying on transmission of the fundamental and second harmonic beams. The fundamental 335 is directed onto a sample cell 345 and the second harmonic waves are generated at the top surface - this surface can be derivatized with immobilized probes or with adsorbed particles, liposomes, cells, etc. The second harmonic waves 340 are generated within a volume element denoted by the circle at the interface between the top surface and the medium contained within cell.

Fig. 8C is a schematic of a configuration substantially similar to the one depicted in Fig. 2 A except that the bottom surface of the sample cell 3, rather than the top, is used to generate the second harmonic waves.

TOTAL INTERNAL REFLECTION

Fig. 9A is a schematic of a waveguide 4 capable of acting as a total internal reflection waveguide which refracts the fundamental 365 and directs it to a location at the interface between the waveguide 380 and a sample cell 375. At this location, denoted by the circle, the fundamental will generate the second harmonic waves and undergo total internal reflection; the second harmonic beam will propagate substantially colinearly with the fundamental and exit the prism 380. Waveguide 380 will typically be in contact with air. In this illustration, the waveguide 380 is a Dove prism. Fig. 9B is a schematic of a configuration similar to the one depicted in Fig. 9A except that the waveguide 400 allows for multiple points of total internal reflection between the waveguide 4 and the sample cell 395, increasing the amount of second harmonic light generated from the fundamental beam.

FIBER OPTIC Fig 10 depicts various configurations of a fiber optic means of delivering or collecting the fundamental or second harmonic beams. In Fig. 10A, the coupling element 410 between a source of the fundamental wave and the fiber optic is depicted. The fundamental, thus coupled into the fiber optic waveguide 405, proceeds to a sample cell 415. In Fig. 10 A, the tip of the fiber can serve as the interface of interest capable of generating second harmonic waves, or the tip can serve merely to introduce the fundamental beam to the sample cell containing suspended cells, particles, etc. In Fig. 10A, the second harmonic light is collected back through the fiber optic.

Fig. 10B is identical to Fig. 10A except that a bead is attached to the tip of the fiber optic (according to means well known in the art). The bead can serve to both improve collection efficiency of the second harmonic light or be derivatized with probes or adsorbed species and presenting an interface with the medium of sample cell 425 capable of generating the second harmonic light.

Fig. IOC is identical to both Figs. 10A and 10B except that collection of the second harmonic light is effected using a solid-angle detector 450. OPTICAL RESONANCE CAVITY

An optical resonance cavity is defined between at least two reflective elements and has an intracavity light beam along an intracavity beam path. The optical cavity or resonator consists of two or more mirrored surfaces arranged so that the incident light can be trapped bouncing back and forth between the mirrors, hi this way, the light inside the cavity can be many orders of magnitude more intense than the incident light. This phenomenon is well known and has been exploited in various ways (see, for example, Yariv A. "Introduction to Optical Electronics", 2^nd Ed., Holt, Reinhart and Winston, NY 1976, Chapter 8). The sample cell can be present in the optical cavity or it can be outside the optical resonance cavity. Fig. 11 is a schematic of an optical resonance power build-up cavity configuration.

Fig. 11 A is a schematic of an optical resonance cavity in which the sample cell 465 is positioned intracavity and the fundamental and second harmonic beams are transmitted through it - a useful configuration for sample cells containing suspended particles, cells, beads, etc. The fundamental beam 455 enters the optical resonance cavity at reflective optic 460 and builds up in power between reflective elements 460 and 462 (intracavity beam). Mirror 460 is preferably tilted (not perpendicular to the direction of the incident fundamental 455) to prevent direct reflection of the intracavity beam back into the light source. The natural reflectivity and transmisivity of 460 and 462 can be adjusted so that the fundamental builds up to a convenient level of power within the cavity. The fundamental generates second harmonic light in a volume element within the sample cell denoted by the circle. Reflective optic 460 can reflect the fundamental and the second harmomc, while reflective optic 462 will substantially reflect the fundamental but allow the pass-through of the second harmonic beam 475 which is subsequently detected. U.S. Pat. No. 5,432,610 (King et al.) describes a diode-pumped power build-up cavity for chemical sensing and it and the references it makes are hereby incorporated by reference herein.

Fig. 1 IB is a schematic of an optical resonance power build-up cavity configuration in which the fundamental beam 475 enters the optical cavity by reflection from optic 480. A second reflective optic element 482 defines the optical resonance cavity. Element 490 is a waveguide (such as a prism) in contact with the sample cell 485 and allows total internal reflection of the fundamental beam at the interface between the waveguide and sample cell surfaces, generating the second harmonic light. Element 482 substantially reflects the fundamental beam but passes through the second harmonic beam 495 which is subsequently detected.

REFLECTION

Fig. 12 A is a schematic of a configuration involving reflection of the fundamental and second harmonic beams. A substrate 525 is coated with a thin layer of a reflective material 520, such as a metal, and on top of this is deposited at layer 515 suitable for attachment of the probes or adsorption of particles, cells, etc. (e.g., SiO₂). This layer is in contact with the sample cell 510. The fundamental 500 passes through the sample cell 510 and generates a second harmonic wave at the interface between layers 515 and 520. The fundamental and second harmonic waves 505 are reflected back from the surface of layer 520.

Fig. 12B is substantially similar to Fig. 11 A except that the second harmonic and fundamental beams are reflected 535 from the interface between the medium contained in sample cell 540 and layer 545. Layer 545 is reflective or partly reflective layer deposited on substrate 550 and is suitable for adsorption of particles, cells, etc. or attachment of probes.

Fig. 12C is a schematic illustrating that only the sample cell 565 need be used for a reflective geometry. The sample cell 565 is partly filled with some medium 570 and the fundamental and second harmomc beams are reflected 560 from the gas-liquid or vapor- liquid interface at the surface of 570. Modes of detection Charge-coupled detectors (CCD) array detectors can be particularly useful when information is desired as a function of substrate location (x,y). CCDs comprise an array of pixels (i.e., photodiodes), each pixel of which can independently measuring light impinging on it. For a given apparatus geometry, nonlinear light arising from a particular substrate location (x,y) can be determined by measuring the intensity of nonlinear light impinging on a CCD array location (Q,R) some distance from the substrate - this can be determined because of the coherent, collimated (and generally co-propagating with the fundamental) nonlinear optical beam) compared with the spontaneous, stochastic and multidirectional nature of fluorescence emission. With a CCD array, one or more array elements {Q,R} in the detector will map to specific regions of a substrate surface, allowing for easy determination of information as a function of substrate location (x,y). Photodiode detector and photomultiplier tubes (PMTs), avalanche photodiodes, phototransistors, vacuum photodiodes or other detectors known in the art for converting incident light to an electrical signal (i.e., current, voltage, etc.) can also be used to detect light intensities. For CCD detector, the CCD communicates with and is controlled by a data acquisition board installed in the apparatus computer. The data acquisition board can be of the type that is well known in the art such as a CIO-DAS16/Jr manufactured by Computer Boards Inc. The data acquisition board and CCD subsystem, for example, can operate in the following manner. The data acquisition board controls the CCD integration period by sending a clock signal to the CCD subsystem. In one embodiment, the CCD subsystem sets the CCD intregration period at 4096 clock periods. By changing the clock rate, the actual time in which the CCD integrates data can be manipulated. During an integration period, each photodiode accumulates a charge proportional to the amount of light that reaches it. Upon termination of the integration period, the charge is transferred to the CCD's shift registers and a new integration period commences. The shift registers store the charges as voltages which represent the light pattern incident on the CCD array. The voltages are then trasmitted at the clock rate to the data acquisition board, where they are digitized and stored in the computer's memory. In this manner, a strip of the sample is imaged during each integration period. Thereafter, a subsequent row is integrated until the sample is completely scanned. Sample substrates and sample cells

Sample substrates and cells can take a variety of forms drawing from, but not limited to, one or more of the following characteristics: fully sealed, sealed or unsealed and connected to flow cells and pumps, integrated substrates with a total internal reflection prism allowing for evanescent generation of the nonlinear beam, integrated substrates with a resonant cavity for fundamental power build-up, an optical set-up allowing for multiple passes of the fundamental for increased nonlinear response, sample cells containing suspended biological cells, particles, beads, etc. Data analysis

Data analysis operates on the vectors of information measured by the detector. The information can be time-dependent and kinetic. It can be dependent on the concentration of one or more biological components, probes, targets, inhibitors, antagonists, agonists, drugs, small molecules, etc. which can be changed during a measurement or between measurements. It can also be dependent on wavelength, etc. In general, the intensity of nonlinear light will be transformed into a concentration or amount of a particular state (for example, the surface-associated concentration of a component or the amount of opened or closed ion-channels in cell membranes), hi one example, the production of second harmonic light follows the equation:

(Is_H)⁰-⁵ E_2ω = Aχ⁽²⁾ + BΦoχ⁽³⁾ (1)

where I_SH is the intensity of the second harmonic light, E _ω is the electric-field amplitude of the second harmonic light, A and B are constants specific to a given interface and sample geometry, Φ₀ is the electric surface potential, and yj ' and γ ³' are the second and third-order nonlinear susceptibility tensors. χ⁽²⁾ is proportional to N (surface-bound or probe-bound targets) and the hypeφolarizability per target. Surface binding reactions can follow a Langmuir-type equation: dN/dt = kι(C-N)/55.5 * (N_maχ-N) - k_ιN (2)

with N the amount of the targets binding to the surface (e.g., targets binding to probes), Nm_aχ the maximum number of the binding species at the surface at equilibrium, ki the association rate constant, k.ι the dissociation rate constant, dN/dt the instantaneous rate of change of the amount of surface-bound targets and C the bulk concentration of the species. Modified Langmuir equations or other equations used in determining the amount of surface-adsorbed or surface-bound species in the art can also be used in the data analysis.

The details of the data analysis will depend on each specific case. If the polarization response due to a net charge on the surface - χ⁽³⁾ - is present, it can be subtracted out in making the measurement. Thus, the number of surface-bound species N can be directly calculated from the second harmomc intensity in this manner. Kinetics or equilibrium properties can be determined from N (at equilibrium or in real time) according, for example, to equation 2 and procedures well known in the art. There are a number of relevant papers in the art which describe this process including, for example: J.S. Salafsky, K.B. Eisenthal, "Second Harmonic Spectroscopy: Detection and Orientation of Molecules at a Biomembrane Interface", Chemical Physics Letters 2000, 319, 435 and Eisenthal, K.B. " Photochemistry and Photophysics of Liquid Interfaces by Second Harmonic Spectroscopy" J. Phys. Chem. 1996, 100, 12997. For probe-target processes which result directly or indirectly in changes in surface charge density or potential (an example of the indirect type is in ion-channel experiments with ion channels in a cell, where a target binds to a probe, leading to the modulation of an ion channel's dynamics which leads, in turn, to the surface charge density). In this case, labels attached to the surface of a cell are used to sense the ion channel's dynamics or state via the effect the surface charge density has on the nonlinear properties of the labels.

6. MISCELLANEOUS

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

All references cited herein are incoφorated herein by reference in their entirety and for all puφoses to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incoφorated by reference in its entirety for all puφoses.

The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

Many modifications and variations of the present invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the invention is to be limited only by the terms of the appended claims along with the full scope of equivalents to which such claims are entitled.

7. REFERENCES

U.S. Patent Documents 5,318,023 June, 1994 Vari et al.

5,408,996 April, 1995 Salb et al. 5,650,135 July, 1997 Contag et al. 5,677,199 October, 1997 Arrhenuis 5,807,261 September, 1998 Benaron et al. 6,167,297 December, 2000 Benaron et al. 6,217,847 April, 2001 Contag et al.

6,246,901 June, 2001 Benaron

6,284,223 September, 2001 Luiken 6,217,847 April, 2001 Contag et al. 6,280,386 Aug., 2001 Alfano et al. 6,208,886 Mar., 2001 Alfano et al. 5,853,370 Dec, 1998 Chance et al.

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Claims

1. A method for imaging a living animal or living cell using a surface-selective nonlinear optical technique, said method comprising the steps of: a) administering to a living animal or living cell one or more selected from the group consisting of a targeting construct, a label, a decorator, an indicator and an enhancer; b) illuminating said living animal or living cell with one or more light beams at one or more fundamental frequencies; and c) detecting nonlinear optical light beam emanating from said living animal or living cell, wherein said nonlinear optical light beam forms an image of said living animal or living cell.

2. The method of claim 1, wherein said method is used to diagnose, identify or treat said tissues, organs, cells or samples which are in a disease state.

3. The method of claim 1, wherein said method is used in a medical procedure, a medical diagnostic procedure or for therapeutic puφoses.

4. The method of claim 1, wherein said imaging method is used in-situ in an animal.

5. The method of claim 1, wherein said imaging method is used in-situ in a mammal.

6. The method of claim 1 , wherein said imaging method is used to image biological tissues, cells or samples that have been obtained from a biopsy procedure.

7. The method of claim 1 , wherein said imaging method is an optical biopsy.

8. The method of claim 1, wherein said imaging method comprises a washing step to remove unbound or non-specifically bound labels, decorators, indicators or enhancers.

9. The method of claim 1 , wherein said living animal or living cell is a cell, a tissue or an organ.

10. The method of claim 1 wherein said nonlinear optical light beam is a second harmonic light beam.

11. The method of claim 1 , wherein said one or more illuminating light beams comprise two light beams with appreciably different frequencies, and the nonlinear optical light beam is the sum frequency, with said sum frequency having a frequency approximately equal to the sum of the frequencies of said two light beams.

12. The method of claim 1, wherein an enhancer is administered, and wherein said enhancer is covalently attached, non-covalently attached, electrostatically attached, chemisorbed or physisorbed to a molecule.

13. The method of claim 1, wherein a label is administered, and wherein said label is covalently attached, non-covalently attached, electrostatically attached, chemisorbed or physisorbed to a molecule.

14. The method of claim 1, wherein a decorator is administered, and wherein said decorator is covalently attached, non-covalently attached, electrostatically attached, chemisorbed or physisorbed to a molecule.

15. The method of claim 1, wherein an indicator is administered, and wherein said indicator is covalently attached, non-covalently attached, electrostatically attached, chemisorbed or physisorbed to a molecule.

16. The method of claim 1, wherein two or more of said labels, decorators, indicators or resonance enhancers with mutually different chemical identities are used in said imaging method.

17. The method of claim 1 wherein said enhancer is attached to or placed in close proximity to a label, decorator or indicator.

18. The method of claim 1 wherein said enhancer is a metal or a metallic particle, a metal-coated particle, a nanoparticle, a colloidal particle, or an aggregate or cluster thereof.

19. The method of claim 1 wherein said label or decorator possesses a chemical or immunological specificity or affinity for a biological component of said living animal or living cell to be imaged by said method.

20. The method of claim 1, wherein the surface-selective nonlinear optical technique is selected from the group consisting of second harmonic, sum frequency or difference frequency generation.

21. The method of claim 1, wherein illuminating the living animal or living cell or detecting the nonlinear optical light beam comprises using one or more modes selected from the group consisting of reflection, transmission, evanescent wave, multiple internal reflection, near-field optical techniques, confocal, optical cavity, planar waveguide, fiberoptic and dielectric-slab waveguide, near-field techniques.

22. The method of claim 1, wherein said detecting comprises measuring one or more physical properties of the nonlinear optical light beam emanating from said living animal or living cell.

23. The method of claim 22, wherein said imaging method further comprises comparing the value of said one or more physical properties of the nonlinear optical light beam measured at a region of said living animal or living cell to a value for said one or more physical properties of the nonlinear optical light beam measured in the absence of said label, decorator, indicator or enhancer.

24. The method of claim 1, wherein said imaging method comprises measuring the intensity of the nonlinear optical light beam at a region or plurality of regions of a living animal or living cell over a period of time.

25. The method of claim 1, wherein said surface-selective nonlinear optical technique comprises a scanning or microscopy technique.

26. The method of claim 1 wherein said imaging method further comprises measuring a background nonlinear optical light beam in the absence of said label, decorator, indicator or enhancer.

27. The method of 1, wherein said optical technique determines the nonlinear light intensity by measuring the intensity of the nonlinear light at a region or plurality of regions with varying target concentration.

28. The method of claim 1 wherein said living animal or living cell is chemically derivatized.

29. The method of claim 1, wherein said imaging method further comprises administering an agonists, an antagonists, a drags, a small molecule or a probe to said living animal or living cell before or during said step of detecting.

30. The method of claim 1, wherein said imaging method further comprises administering an inhibitor before or during said step of detecting.

31. The method of claim 1, wherein the one or more illuminating light beams or the nonlinear optical light beam is linearly or circularly polarized.

32. The method of claim 1, wherein said imaging method further comprises measuring one or more physical properties of the nonlinear optical light beam, said one or more physical properties selected from the group consisting of (i) the intensity of the nonlinear optical light beam, (ii) the wavelength or spectrum of the nonlinear optical light beam, or (iii) the time-course of (i) or (ii).

33. The method of claim 1, wherein said imaging method further comprises the use of a fiber optic line for delivery of the illuminating light or collecting the nonlinear optical light.

34. The method of claim 1, wherein said imaging method is an optical biopsy or a minimally invasive in-vivo or in-situ optical imaging method.

35. The method of claim 1, wherein said administering step comprises introducing a nucleic acid encoding a fusion protein comprising a label such that said fusion protein is expressed by said living animal or living cell.

36. The method of claim 35, wherein said fusion protein comprises bacteriorhodopsin, green fluorescent protein, or a genetic mutant thereof.

37. The method of claim 1, wherein the fundamental frequencies of said one or more illuminating light beams or a frequency of said said one or more nonlinear optical light beam corresponds to wavelengths in the range of 100 nanometers to 3000 nanometers.

38. A method of measuring a target site within a living subject, comprising: a) administering to the living subject an nonlinear-active contrast agent selected so as to provide a nonlinear optical signal contrast for said target site in vivo when said target site is illuminated with a light source; b) allowing said contrast agent to achieve sufficient distribution and localization within the body of said living subject; c) using a medical device or instrument in the performance of a medical or surgical procedure upon said subject, wherein said medical device or instrument is optically coupled to a light source and a light detector; d) illuminating the target site with light from the light source, said light source selected such that the contrast agent in vivo may interact with said light and generate a nonlinear optical signal; e) detecting said nonlinear optical signal using a light detector; f) determining a measurable parameter of the target site using the detected light based upon a function of the distribution and localization of said contrast agent; and, g) generating an output signal using said measurable parameter.

39. A method for in vivo identification of tumor tissue associated with a disease state in a living subject, said method comprising: a) administering to a living subject having directly viewable tumor tissue a diagnostically effective amount of at least one biologically compatible nonlinear-active targeting construct comprising a tumor- avid moiety, so as to allow the nonlinear-active targeting construct to bind to and/or be taken up selectively in vivo by the directly viewable tumor tissue; b) illuminating an in vivo body part of the living subject comprising the directly viewable tumor tissue with a light beam having at least one wavelength in the nonlinear optical response spectrum of the nonlinear-active targeting construct; and c) detecting a nonlinear optical light beam emanating from any targeting construct bound and/to or taken up by the directly viewable tumor tissue, wherein the nonlinear optical light beam indicates the location and/or surface area of the tumor tissue in the in vivo body part.